Intracellular sorting and transport of proteins

Progress in Biophysics & Molecular Biology 83 (2003) 1–45

Review

Intracellular sorting and transport of proteins

Catherine van Vlieta, Elaine C. Thomasb, Ana Merino-Trigoa,Rohan D. Teasdaleb, Paul A. Gleesona,*

aThe Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Melbourne,

Vic. 3010, Australiab Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld 4072, Australia

Abstract

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, eachwith a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules todefined locations and to ensure that the identity, and hence function, of individual compartments aremaintained. The localisation of proteins to specific membranes is complex and involves multipleinteractions. The recent dramatic advances in understanding the molecular mechanisms of membranetransport has been due to the application of a multi-disciplinary approach, intergrating membrane biology,genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is tosummarise the general principles of protein sorting in the secretory and endocytic pathways and tohighlight the dynamic nature of these processes. The molecular mechanisms involved in this transport alongthe secretory and endocytic pathways are discussed along with the signals responsible for targeting proteinsto different intracellular locations.r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Protein sorting; Membrane transport; Golgi apparatus; Endosomes

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Components of the secretory pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1. The endoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1.1. Glycosylation in the ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.2. The ER-Golgi intermediate compartment . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.3. The Golgi apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

*Corresponding author. Tel.: +61-3-8344-5912; fax: +61-3-9347-7730.

E-mail address: [email protected] (P.A. Gleeson).

0079-6107/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.

doi:10.1016/S0079-6107(03)00019-1

1. Introduction

Eukaryotic cells are highly compartmentalised into distinct membrane-bound organelles, afeature essential for fundamental as well as specialised cellular processes. To function, eachmembrane-bound organelle has a unique composition of proteins and lipids. Therefore, it is notsurprising that highly specific transport mechanisms are required to direct molecules to definedlocations and to ensure that the identity, and hence function, of individual compartments aremaintained. Proteins contain structural information that targets them to their correct destinationand many targeting signals have now been defined. The organelles of the secretory pathway are

2.3.1. Glycosylation in the Golgi apparatus . . . . . . . . . . . . . . . . . . . . . . . 7

2.4. The trans-Golgi network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2.5. The cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3. Transport in the secretory pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3.1. Transport from the ER to the Golgi . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3.2. Transport through the Golgi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.2.1. Anterograde vesicular transport . . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.2.2. Cisternal progression/maturation . . . . . . . . . . . . . . . . . . . . . . . . . 10

3.2.3. Dual-transport model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3.3. Transport from the TGN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

4. Molecular mechanisms of vesicular transport . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.1. Coat proteins and vesicle biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.1.1. COPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.1.2. COPII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

4.1.3. Clathrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

4.1.4. Adaptor protein complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4.1.5. Adaptor-related proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4.2. Vesicle docking and fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.2.1. SNAREs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.2.2. Rabs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4.2.3. Tethers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

5. Localisation signals in the secretory pathway . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5.1. ER retention and retrieval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5.2. Golgi localisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

5.2.1. Glycosyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

5.2.2. Peripheral Golgi membrane proteins . . . . . . . . . . . . . . . . . . . . . . . 25

5.3. Targeting to apical and basolateral membranes in polarised cells . . . . . . . . . . . . 26

6. Endosome protein sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

6.1. Dissection of endosomal sorting in yeast . . . . . . . . . . . . . . . . . . . . . . . . . 28

6.2. Human retromer complex and sorting nexins . . . . . . . . . . . . . . . . . . . . . . 30

7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

C. van Vliet et al. / Progress in Biophysics & Molecular Biology 83 (2003) 1–452

involved in the sorting of proteins to a variety of intracellular membrane compartments and thecell surface (Fig. 1). For example, proteins that are transported within the secretory pathway areeither secreted from the cell, bound to the plasma membrane, sorted to lysosomes, or are retainedas ‘‘residents’’ in any of the organelles. Within the endoplasmic reticulum (ER), newly synthesisedproteins are scrutinised to ensure they are correctly folded, undergo initial glycosylation beforebeing packaged into transport intermediates or vesicles, and then moved forward to the entry siteof the Golgi apparatus. Secretory proteins are then transported through the Golgi cisternae to thetrans-Golgi network (TGN), or Golgi exit site. At the TGN proteins are sorted according to theirfinal destinations. The TGN is also the site where the biosynthetic and endocytic pathways

Fig. 1. The secretory and endocytic pathways. Newly synthesised membrane and soluble proteins that reside in the

secretory and endocytic pathways, as well as proteins that are destined for secretion, are translocated into the

endoplasmic reticulum (ER), then packaged into COPII coated vesicles which fuse to become the ER-Golgi

intermediate compartment (ERGIC). Numerous ERGICs merge to form the cis-Golgi network (CGN). COPI coated

vesicles recycle ER proteins to and from the ERGIC and Golgi stack. Anterograde cargo moves through the Golgi

stack and is sorted according to destination at the trans-Golgi network (TGN). Different types of coated vesicles and

tubulovesicular carriers transport cargo to various destinations. Proteins are endocytosed at the plasma membrane and

transported to the early endosome. From the early endosome proteins can be recycled to the cell surface, transported to

the lysosome via the late endosome, or to the TGN.

C. van Vliet et al. / Progress in Biophysics & Molecular Biology 83 (2003) 1–45 3

converge. Molecules are internalised from the cell surface in endocytic vesicles and transported tothe early endosome where extensive sorting takes place. For example, endocytosed proteins canthen be recycled to the plasma membrane (such as recycling receptors), or transported to the TGNor to the lysosome via the late endosomes for degradation. Thus, the TGN and the earlyendosome represent the two major sorting stations of the cell.

Protein transport in the secretory and endocytic pathways is a multi-step process involving thegeneration of transport carriers loaded with defined sets of cargo, the shipment of the cargo-loaded transport carriers between compartments, and the specific fusion of these transportcarriers with a target membrane. These processes involve a complex interplay of protein and lipidinteractions. Over the past 10–15 years much of the cellular machinery responsible forintracellular transport and protein sorting has been described. Significantly, with the identificationof many genes that cause disease, it is now clear that a number of human diseases are due todefects of intracellular trafficking (Aridor and Hannan, 2000; Olkkonen and Ikonen, 2000),emphasising the biological importance of these dynamic membrane-mediated processes.

The field of membrane transport and protein sorting integrates membrane biology, genetics,imaging, protein and lipid biochemistry and structural biology. For the newcomer it can be adifficult area to obtain a broad overview. The aim of this review is to summarise the generalprinciples of protein sorting in the secretory and endocytic pathways and in particular, highlightthe dynamic nature of these processes. The molecular mechanisms involved in this transport alongthe secretory pathway is discussed together with the signals responsible for targeting proteins todifferent intracellular locations. Finally, the recent emerging details of the molecular machineryassociated with endosome sorting is reviewed.

2. Components of the secretory pathway

2.1. The endoplasmic reticulum

Protein translation begins on free ribosomes in the cytosol. Proteins destined for secretion orfor residence along the secretory pathway are targeted to the ER lumen by an N-terminal signalsequence (Walter and Johnson, 1994). As the nascent polypeptide chain emerges from freeribosomes it is bound by the signal recognition particle (SRP) that directs the ribosome andnascent chain to the ER membrane. The ribosome-nascent chain-SRP complex then binds to thecytosolic side of the ER membrane via interactions with the Sec61p complex and SRP receptor(Rapoport et al., 1996; Matlack et al., 1998). The polypeptide chain is cotranslationallytranslocated into the lumen of the ER through the Sec61p membrane channel, referred to as thetranslocon, with the assistance of the translocating-chain associating membrane (TRAM) protein(Gorlich and Rapoport, 1993). As the nascent chain is translocated into the lumen of the ER theN-terminal signal sequence of soluble proteins is cleaved by signal peptidase (Martoglio andDobberstein, 1998). Transmembrane proteins are cotranslationally inserted into the membrane ofthe ER (Do et al., 1996).

Within the lumen of the ER are a number of chaperones that bind to the polypeptide chain andassist the protein in forming the correct conformation. These chaperones include BiP (Gething,1999), the lectins calnexin and calreticulin (Helenius et al., 1997) and protein disulphide isomerase


(Freedman, 1994). Chaperones assist in the folding of the nascent polypeptide chain protein byslowing folding, preventing aggregation and ensuring the correct disulphide bonds are formed.The chaperone proteins also have a role in ‘‘quality control’’, directing malformed proteins backthrough the translocon and to the proteosome for degradation (Lord et al., 2000).

2.1.1. Glycosylation in the ER

The majority of plasma membrane and secretory proteins are glycosylated. N-linkedoligosaccharides are added to the growing polypeptide chain as it enters the ER (Kornfeld andKornfeld, 1985). A presynthesised 14-residue oligosaccharide (Glc3Man9GlcNAc2) is transferredfrom a dolicol-lipid carrier to an asparagine residue in the consensus sequence -N-X-S/T- (whereX is any amino acid except proline) in the nascent polypeptide (Schachter et al., 1983; Kornfeldand Kornfeld, 1985). This reaction is catalysed by an oligosaccharyl-transferase. Once linked tothe protein two of the glucose residues are removed by glucosidases I and II and one mannose isremoved by an ER-a-mannosidase, resulting in GlcMan8GlcNAc2 (Helenius et al., 1997). Thistrimmed oligosaccharide plays an important role in ensuring the correct folding of N-linkedglycoproteins. The monoglucosylated form is bound by the chaperones calnexin or calretriculin,which assists the folding process (Hammond and Helenius, 1995). Glucosidase II hydrolyses theremaining glucose residue from the monoglucosylated species, causing the chaperones todissociate (Helenius, 1994). If the newly formed glycoprotein is incorrectly folded, it isreglucosylated by UPD-Glc:glycoprotein glucosyltransferase and again recognised by calnexinand/or calreticulin (Trombetta and Parodi, 1992; Parodi, 2000). This cycle allows the protein toform the correct conformation and also assists in the removal of misfolded proteins. Oncecorrectly folded the protein is ready to exit the ER in vesicles budding from ER exit sites, ortransitional elements (Palade, 1975). N-linked oligosaccharides leave the ER as Man8GlcNAc2moieties and are generally modified further as they are transported through the Golgi.

O-linked glycosylation begins in the ER or cis-Golgi where a GalNAc residue is added to aserine or threonine residue by polypeptide N-acetylgalactosaminyl transferase (Marth, 1996).Other sugar residues are often added to the GalNAc-Ser (Thr) in the Golgi, but generally theoligosaccharide chains are short, between 1 and 4 residues long (Brockhausen, 1995).

2.2. The ER-Golgi intermediate compartment

Proteins are exported from the ER at specialised exit sites called transitional elements ortransitional ER (tER) (Palade, 1975). The tER are smooth ribosome-free sections of the ERadjacent to the rough ER. The tER are located close to the central cis-Golgi area but are alsofound throughout the periphery of the cell (Klumperman, 2000). The tER are specialised domainsfor the production of transport vesicles. tER-derived transport vesicles subsequently fuse to forma network of vesicular tubular clusters (VTCs) (Bannykh et al., 1996), also known as pre-Golgiintermediates (Saraste and Kuismanen, 1992) or the ER-Golgi intermediate compartment(ERGIC) (Hauri and Schweizer, 1992). The ERGIC was identified as a compartment in whichcargo accumulates during a block in traffic from the ER to the Golgi at 15�C (Saraste andKuismanen, 1984; Schweizer et al., 1990). The ERGIC compartment is a major sorting station,recycling ER proteins in retrograde vesicles as well as delivering secretory cargo to the cis-Golgi(Warren and Mellman, 1999). Peripheral ERGICs move along microtubules to the Golgi region


(Presley et al., 1997; Scales et al., 1997) where they fuse to form the cis-Golgi network, (Sarasteand Kuismanen, 1992; Presley et al., 1997), a compartment which contains the cis most Golgicisternae (Ladinsky et al., 1999).

2.3. The Golgi apparatus

The Golgi apparatus was first discovered just over a century ago. Italian biologist Camilo Golgideveloped a specialised staining technique using heavy metal impregnation, called the ‘blackreaction’, to study neuronal cells (reviewed by Berger, 1998). In 1906 Camillo Golgi shared theNobel Prize for Physiology for his work in developing this stain and his contribution to theunderstanding of neuronal networks. Golgi is better known, however, for the discovery ofthe ‘apparato reticulare interno’ in 1898 (Golgi, 1898). Using the ‘black reaction’ on Purkinjecells, Golgi stained an intricate tubular network surrounding the nucleus, a structure known todayas the Golgi apparatus, the Golgi complex or simply the Golgi. Fifty years of controversy ensuedas to whether the Golgi represented a real structure or was an artefact of the staining method(Farquhar and Palade, 1981). The controversy was not resolved until the first electronmicrographs of the Golgi were published in 1954 that showed a membranous network in theregion where the Golgi was detected by the ‘black reaction’ (Dalton and Felix, 1954; Sj .ostrandand Hanzon, 1954). These studies described the Golgi as a stack of flattened cisternae surroundedby vesicles and vacuoles of various sizes. Subsequent studies in the 1960s and 1970s using the newtechniques of pulse chase and autoradiography, revealed the role of the Golgi apparatus in theglycosylation of proteins and as a central component of the secretory pathway (Neutra andLeblond, 1966; Palade, 1975).

The Golgi apparatus consists of a series of flattened membrane cisternae, called the Golgi stack,bordered by two tubulo-vesicular networks, the cis-Golgi network (CGN) and the trans-Golginetwork (TGN). Long membrane tubules interconnect multiple Golgi stacks, which are arrangedaround the nucleus, close to the centrosome (Thyberg and Moskalewski, 1999). The cis face of theGolgi receives cargo from the ER, which is transported through the Golgi stack to the TGN. TheCGN is likely to arise from the fusion of several ERGIC elements and, as such, assists in sortingsecretory cargo from ER residents as described in the previous section. The TGN sorts andpackages proteins into membrane carriers destined for the plasma membrane, endosomes andregulated secretory granules (Keller and Simons, 1997; Traub and Kornfeld, 1997), and isdiscussed in more detail in the next section.

The number of cisternae in a Golgi stack varies between species and cell types but generallybetween 3 and 8 cisternae are observed (Farquhar, 1985). The stack can be divided into threemorphologically and functionally distinct sub-compartments, the cis-, medial- and trans-Golgi.The heterogeneity of these cisternae was first illustrated by differential staining for variousenzymes (reviewed by Farquhar and Palade, 1981). Novikoff and Goldfischer (1961)demonstrated that the enzymes thiamine pyrophospatase and nucleoside diphosphatase wererestricted to the trans-Golgi, Friend and Murray (1965) found that osmium impregnationpreferentially stained the cis-Golgi. It was discovered some time later that there is also a gradientof cholesterol concentration across the stack (Orci et al., 1981).

Further evidence of compartmentalisation of the Golgi stack was clearly evident from studieson the location of the glycosylation machinery. Golgi glycosyltransferases were found to have


distributions that reflected the order of glycosylation events (Kornfeld and Kornfeld, 1985;Rabouille et al., 1995a). For example, immunoelectron microscopy revealed that late actingenzymes were localised to the trans-cisternae and TGN (Roth and Berger, 1982). The observationthat the synthesis of complex oligosaccharides follows the order that glycoproteins contactenzymes across the stack suggests that the distribution of glycosylation enzymes provides a levelof control over glycosylation events.

Modifying enzymes other than glycosyltransferases also reside within the Golgi. These includeprocessing a-mannosidases and N-acetylglucosamine-1-phosphodiester-a-N-acetylglucosamini-dase, the latter responsible for the generation of the mannose-6-phosphate (M6P) phospho-mannosyl residue that is recognised by M6P (Pohlmann et al., 1982). M6P receptors directglycoproteins tagged with M6P to lysosomes via endosomes.

The Golgi also plays a role in lipid biosynthesis with increasing concentrations of cholesteroland sphingolipids present across the stack (van Meer, 1998). The early Golgi enzymesphingomyelin synthase produces sphingomyelin and diacylglycerol from phosphatidylcholineand ceramide generated in the ER (Futerman et al., 1990; Jeckel et al., 1990). Higher orderglycosphingolipids are synthesised in the late Golgi (Lannert et al., 1998). Association of ER-derived cholesterol with sphingolipids results in their segregation from unsaturated glycerolipidsin the lipid bilayer (Holthuis et al., 2001). As these sphingolipid/cholesterol domains moveforward through the Golgi, unsaturated glycerolipids are recycled, resulting in a cis–trans

sphingolipid/cholesterol gradient (Holthuis et al., 2001) and increasing bilayer thickness (Neziland Bloom, 1992).

2.3.1. Glycosylation in the Golgi apparatusA large number of glycosidases and glycosyltransferases reside within the Golgi. These

glycosylation enzymes modify the asparagine linked Man8GlcNAc2 structure that arrives from theER. The reactions catalysed by the various combinations of these enzymes produce an incrediblydiverse repertoire of oligosaccharide structures. N-linked glycans fall into three broad categories:high mannose, complex and hybrid glycans (Kornfeld and Kornfeld, 1985). The final glycanstructure depends on multiple factors, including, the protein sequence, organism, cell type,location and activity of the various enzymes as well as availability of substrates (Varki, 1998).

The processing of N-linked oligosaccharides that occurs in the Golgi is well described bySchachter et al. (1983) and Kornfeld and Kornfeld (1985). If an N-linked Man8GlcNAc2 chain isuntrimmed or only partially trimmed to Man5GlcNAc2 by mannosidase I (Man I) in the cis-Golgi, a high mannose structure results. Alternatively this structure can undergo furthermodification in the medial-Golgi. N-acetylglucosaminyl transferase I (GlcNAc-TI) adds a N-acetylglucosamine (GlcNAc) to the glycan chain. The product of this reaction can then go on toform a complex or hybrid glycan. A hybrid glycan is produced by the addition of GlcNAc byGlcNAc-TIII to the mannose residue at the base of the branch, creating a ‘bisected’ glycan thatcannot be processed further by the Golgi mannosidase, Man II. Alternatively, a complex glycan isformed by removal of two mannose residues by Man II, followed by addition of another GlcNAcby GlcNAc-TII, resulting in a two-branch glycan with outer chain GlcNAc residues. Thesebranches can be extended by addition of fucose, galactose and sialic acid by multiplefucosyltransferases, galactosyltransferases and sialyltransferases, respectively.


The presence of a particular glycosyltransferase is often used as a marker for a particularcompartment, for example, GlcNAc-TI as a medial-Golgi enzyme and a2; 6-sialyltransferase as aTGN marker. These enzymes, however, are not restricted to a single cisterna but have overlappingdistributions (Rabouille et al., 1995a).

2.4. The trans-Golgi network

The TGN is functionally and morphologically distinct from the Golgi stack. Several featuresdifferentiate the TGN from the stack cisternae (Griffiths and Simons, 1986). The TGN is atubular reticular network and has coated buds whose coats differ from those found in the Golgistack (Schmid, 1997; Boman, 2001). There are a number of distinct processing activities that arerestricted to the TGN. These include: sialylation (Chege and Pfeffer, 1990), proteolytic processing(Molloy et al., 1994), tyrosine sulphation (Baeuerle and Huttner, 1987). There is also an increasingnumber of proteins identified that are specifically localised to the TGN, including: TGN38 (Luzioet al., 1990); p200 (Narula and Stow, 1995); p230 (Gleeson et al., 1996); the adaptor proteins, AP1and AP3 (Robinson and Bonifacino, 2001) and adaptor related proteins, the GGAs (Boman,2001).

The TGN is a highly dynamic compartment involved in sorting of cargo for delivery to multipledestinations (Fig. 1). Proteins are packaged into membrane carriers for transport to the apical andbasolateral membranes in polarised cells, regulated secretory granules and the endosome/lysosome system (Keller and Simons, 1997; Traub and Kornfeld, 1997). Proteins can also berecycled from the TGN to earlier compartments in the secretory pathway (Cole et al., 1998).

2.5. The cytoskeleton

The cytoskeleton plays an important role both in the maintenance of organelle location withinthe cell and in the efficient transport of vesicles throughout the cell (Lippincott-Schwartz, 1998).The cytoskeleton provides filamentous tracks along which membrane transport carriers areguided as they move from one compartment to another. The primary cytoskeletons of mammaliancells are composed of microtubules, actin and intermediate filaments; both microtubules and actinhave important roles in the secretory and endocytic pathways.

Microtubules radiate from a perinuclear microtubule organising centre (MTOC). They arepolarised, with their plus-ends located at the cell periphery and minus-ends at the MTOC. TheGolgi apparatus is localised adjacent to the MTOC through association with the minus ends ofmicrotubules (Cole and Lippincott-Schwartz, 1995). Efficient long distance transport within thecell is dependent on microtubules (Kamal and Goldstein, 2000). For example, transport carriersleaving the ER travel towards the Golgi on microtubular tracks (Presley et al., 1997; Scales et al.,1997). Membrane carriers between the Golgi and plasma membrane also utilise microtubules(Toomre et al., 1999).

The actin cytoskeleton also plays a role in membrane trafficking and recent data indicates thatsome membrane carriers utilise both microtubules and actin cytoskeletons in a single journey(Kelleher and Titus, 1998; Goode et al., 2000). A number of motor proteins including themicrotubule associated motors, dynein and kinesin, and the actin motor, myosin, associate eitherdirectly with cargo, for example, rhodopsin (Tai et al., 1999) or via linker proteins, such as the


CLIPs (see below), which bridge membranes with the cytoskeleton (Goode et al., 2000). Bothactin and microtubule motor proteins, together with linker proteins, are thought to form largecomplexes to facilitate smooth transition of transport carriers between the different cytoskeletons(Goode et al., 2000).

Cytoplasmic linker proteins (CLIPs) mediate interactions between organelles and microtubules(Schroer, 2000). These proteins have long central coiled coil domains capped by non-coiled coilends, which interact with either the membrane or microtubules. The founding member of thisprotein family, CLIP-170, links endosomal membranes to microtubules (Pierre et al., 1992).CLIPs are believed to be involved in maintenance of organelle structure and may also have a rolein regulating vesicle transport by linking membrane carriers to microtubules (Schroer, 2000).GMAP-210 (Golgi microtubule-associated protein of 210kDa), another microtubule bindingprotein with a similar domain structure to the CLIPs, links the cis-Golgi network to microtubules(Rios et al., 1994; Infante et al., 1999). The N-terminus of this protein binds to cis-Golgimembranes, while the C-terminus binds to microtubules (Infante et al., 1999).

3. Transport in the secretory pathway

There are a variety of distinct pathways within the secretory system in which protein and lipidcargo can be transported. Each pathway is highly selective for certain cargo. The transportmechanisms that operate from each compartment reflect the requirement to target cargomolecules to specific destinations and yet at the same time maintain the membrane and proteincomposition of the individual compartments. To maintain the homeostatis of organelles in thesecretory pathway not only protein traffic but also lipid traffic must be tightly regulated, via anappropriate balance of forward and backward membrane transport pathways.

3.1. Transport from the ER to the Golgi

Secretory cargo leaving the ER is packaged into vesicles with a specialised protein coat knownas COP (Coat Protein) II (Barlowe, 1998). Much debate has occurred over the years as to whetherthis transport process is selective or if cargo is transported as part of a bulk flow process. Bulkflow suggests that cargo is simply packaged into vesicles and is concentrated as it moves throughthe Golgi, as a consequence of other proteins being retained within specific compartments orrecycled to upstream compartments (Wieland et al., 1987). In this model, signals on proteins arenecessary for recycling and retention, whereas absence of these signals will lead to secretion.Selective, or receptor mediated, transport predicts that proteins are concentrated during exportfrom the ER by a process involving signal recognition (Kuehn and Schekman, 1997). This lattermodel predicts that cargo molecules contain signal sequences to direct forward transport.

It is likely that cargo leaves the ER both by selective mechanisms and bulk flow: highlyabundant proteins could be transported by bulk flow mechanisms, while rare proteins would bepackaged into COPII vesicles by selective uptake (Warren and Mellman, 1999). Followingbudding from the ER, COPII vesicles lose their coats and fuse to form ERGICs (Aridor et al.,1995). Another COP coat, called COPI, assembles on the membranes of the ERGICs generatingvesicles for retrieval of ER residents (Aridor et al., 1995; Scales et al., 1997). Recent findings


indicate that soluble cargo is concentrated by selective removal of ER proteins in retrograde COPIcoated vesicles (Martinez-Menarguez et al., 1999). The concentration of not only soluble but alsomembrane cargo proteins by exclusion from recycling vesicles is reasonable given thatapproximately 90% of the ERGIC membrane is thought to be recycled to the ER (Warren andMellman, 1999). ERGICs, also known as pre-Golgi intermediates, are highly dynamic structuresand move as discrete entities along microtubules to the cis face of the Golgi, where they fuse toform the CGN de novo (Saraste and Kuismanen, 1992; Lippincott-Schwartz et al., 2000).

3.2. Transport through the Golgi

Since the discovery of protein transport through the Golgi, two alternate models for thisprocess have dominated the field of cell biology, and for many years it was believed they wereirreconcilable. The anterograde vesicular transport model proposed that the individual cisternaeare static compartments and that cargo was transported through the stack in vesicles buddingfrom one cisterna and fusing with the next. On the other hand, cisternal progression predicted thatcargo was transported through the stack in cisternae as they progress through the stack from thecis to trans face. Models comprising elements of both cisternal progression and vesicular transportmay better represent the movement of cargo across the Golgi stack (Pelham and Rothman, 2000).

3.2.1. Anterograde vesicular transportIn the earlier vesicular transport model, the individual Golgi cisternae were seen as static

compartments, with anterograde vesicles transporting cargo progressively from one cisterna to thenext. The concept of static cisternae was able to readily explain the different distributions of Golgienzymes throughout the stack, with each compartment containing a unique set of proteins.Evidence for this model emerged from in vitro transport assays which supported the concept thatcargo was transported from one Golgi compartment to another in small COPI coated vesicles(Orci et al., 1986). COPI coated vesicles were found to bud from Golgi membranes in vitro andsurround Golgi stacks in vivo. Treatments that prevented COPI vesicle formation were found toinhibit anterograde cargo transport (Rothman and Orci, 1992; Chardin and McCormick, 1999).

Difficulties arose with the vesicle transport model when it was discovered through yeast geneticsthat COPI vesicles mediate retrograde transport from the Golgi to the ER (Cosson andLetourneur, 1994; Letourneur et al., 1994). Subsequent studies demonstrated that retrogradetransport operates, not only from the ERGIC back to the ER, but indeed throughout the lengthof the secretory pathway (Rapak et al., 1997; Cole et al., 1998). As COPI coated vesicles are theonly vesicles observed to bud from Golgi stacks and they clearly play a role in retrogradetransport, it has been difficult to imagine how these vesicles could also achieve anterogrademovement of cargo. Studies in yeast also revealed that there are insufficient fusion markers tolabel each compartment separately, which would be necessary for directional vesicular transportbetween multiple cisternae within the Golgi (Pelham, 1998).

3.2.2. Cisternal progression/maturation

Cisternal progression was first proposed in the 1950s, and later refined by Morr!e (1987). Itsuggests that the individual cisternae move progressively through the stack carrying anterogradecargo. Cisternae are formed by fusion of ER-derived membrane structures at the cis face of the


stack, and broken down into various transport intermediates at the trans face. Studies showing de

novo formation of cis-Golgi cisternae provide compelling support for this model (Saraste andKuismanen, 1992; Presley et al., 1997). This model also allows for the transport of large molecularstructures, such as algal scales and procollagen (Melkonian et al., 1991; Bonfanti et al., 1998).

One difficulty with the cisternal progression model was the finding that individualcompartments are not identical but instead contain unique mixtures of proteins (Farquhar andPalade, 1981). As individual glycosyltransferases are concentrated in particular compartmentsand form gradients across the stack, it was hard to envision a cisterna simply moving through thestack without it being somehow altered by both addition and removal of these Golgi residents.The discovery of retrograde transport provided a means to achieve distinct gradients of residentGolgi proteins and the concept of cisternal maturation evolved from the earlier cisternalprogression model (Glick et al., 1997; Pelham, 1998). Selective delivery of resident Golgi enzymesby retrograde transport could result in enrichment of particular glycosyltransferases in specificcompartments. Indeed, several studies have observed increased concentrations of Golgi enzymesin peri-Golgi vesicles suggesting a role for these vesicles in the recycling of Golgi residents (Loveet al., 1998; Lanoix et al., 2001; Martinez-Menarguez et al., 2001). Thus, in the cisternalmaturation model, cisternae ‘mature’ with the selective removal and addition of components asthey progress along the stack.

Additional evidence in support of cisternal maturation is the finding that the anterogrademarker vesicular somatitis virus membrane glycoprotein (VSVG) is largely excluded from peri-Golgi vesicles in vivo (Martinez-Menarguez et al., 2001; Mironov et al., 2001). Furthermore,VSVG and procollagen were found to move through the Golgi at similar rates, supportingcisternal maturation as the mechanism of transport for these proteins (Mironov et al., 2001).

However, one problem with cisternal maturation model is that it cannot explain the origin ofthe TGN, an apparently steady-state compartment, which contains a unique set of residentproteins and behaves differently from the Golgi stack in many respects (Griffiths, 2000).

3.2.3. Dual-transport model

As outlined above, neither Golgi transport model can adequately account for all current data.Therefore, the question of the mechanism of traffic through the Golgi remains debated. Vesiculartransport and cisternal maturation need not be mutually exclusive and may in fact be occurringsimultaneously (Pelham and Rothman, 2000). One proposal is that large molecules and proteinaggregates too large to fit into vesicles could be transported inside maturing cisternae, while bi-directional vesicles could provide a ‘‘fast track’’ for cargo transport through the Golgi (Orci et al.,2000). Electron microscopic data suggests that distinct populations of COPI vesicles, buddingfrom many levels of the Golgi stack, carry either anterograde or retrograde-directed cargo (Orciet al., 1997). Rothman and colleagues have proposed that one population of COPI vesicles,marked by the Golgi SNARE GOS-28, may ‘‘percolate’’ up and down the Golgi stack (Orci et al.,2000). Molecular tethers observed linking COPI vesicles to Golgi membranes would constrain thevesicles, limiting the transfer of cargo to adjacent cisternae in the stack (Orci et al., 1998). Buddingfrom one cisterna and randomly fusing with an adjacent cisterna, these vesicles would result in netflow of cargo through the Golgi as newly synthesised cargo is continually added at the cis face andremoved at the trans face. This ‘‘random walk’’ of vesicles across the Golgi stack would overcomethe requirement for multiple sets of specific SNARE complexes (Orci et al., 2000). Another


population of COPI vesicles would carry retrograde proteins (Pelham and Rothman, 2000). Thismodel would provide the ability to segregate cargo from resident Golgi enzymes into fast and slowmoving transport carriers. It is likely that various transport pathways apply for different proteins.

3.3. Transport from the TGN

Proteins are sorted at the TGN for delivery to multiple destinations including: the basolateraland apical plasma membranes; secretory granules; endosomes; and for retrograde transport(Fig. 1). Specific mechanisms apply to each pathway and these are slowly being unravelled.Mechanisms for sorting to these different locations are quite distinct but are generally signaldependent (Keller and Simons, 1997).

Constitutive secretion was once thought to occur exclusively in small vesicles (Rothman andWieland, 1996; Schekman and Orci, 1996). However, imaging of live cells expressing greenfluorescent protein (GFP) fusion proteins have shown that a variety of membrane cargo istransported to the plasma membrane in large tubulovesicular structures (Hirschberg et al., 1998;Toomre et al., 1999). A difficulty with the use of GFP-fusion proteins is that the typical highlevel expression of GFP-fusions could alter normal trafficking events (Girotti and Banting, 1996;Stephens and Pepperkok, 2001). However, an increasing body of evidence suggests thatlarge pleiomorphic membrane carriers are required for long distance travel, such as from the ERto Golgi and from the TGN to plasma membrane (Lippincott-Schwartz et al., 2000; Stephensand Pepperkok, 2001). It is possible that the conditions in the in vitro assays used to deciphermany of the mechanistic details of membrane transport favour the formation of vesicles, ratherthan larger membrane carriers, which is why the later was only observed with the advent of livecell imaging.

In polarised epithelial cells, plasma membrane proteins and soluble cargo are delivered to eitherthe apical or basolateral surface. Proteins can be delivered directly to the target membrane orindirectly via the opposite membrane (Mostov et al., 2000). In the indirect route, proteins are firstdelivered to one surface and then endocytosed and delivered to early endosomes for transcytosisto the target membrane. Endocytosed proteins are transported from early endosomes to recyclingendosomes, from where they are transported either directly to the basolateral surface or to theapical surface, via the apical sorting endosome (Brown et al., 2000).

Proteins destined for regulated secretion aggregate in the TGN where they are packaged intoimmature secretory granules (ISGs) (Thiele et al., 1997). Clathrin coated vesicles form on theseISGs and recycle TGN proteins, resulting in further concentration of the secretory proteins in amanner analogous to anterograde cargo concentration by removal of ER proteins from ERGICsin COPI vesicles. Mature secretory granules are stored in the cytosol until the cell receives a signalto release their contents. Signalling triggers the transport of secretory granules and their fusionwith the plasma membrane thereby releasing their contents (Burgess and Kelly, 1987).

Lysosomal enzymes are transported from the TGN in specialised vesicles. These vesicles carryenzymes and their receptors to the early/late endosomes. For example, many lysosomal proteinsobtain M6P moieties in the Golgi that are recognised by M6P receptors and packaged into vesicleswith specialised protein coats for delivery to lysosomes via endosomes (Rouille et al., 2000). TheM6P receptors are then recycled from endosomes back to the TGN. For a review on thetrafficking of lysosomal enzymes see Hunziker and Geuze (1996).


4. Molecular mechanisms of vesicular transport

Transport of proteins and lipids within eukaryotic cells requires budding of membrane carriersfrom one compartment (the donor membrane) and fusion with another compartment (theacceptor membrane). To maintain the unique composition of individual organelles and preventinappropriate mixing of compartments, it is essential that this budding and fusion process istightly regulated.

Membrane bound vesicles are required for many transport steps within the secretory andendocytic pathways. While each step utilises different proteins for coat formation, vesicle buddingand fusion, the principles behind these processes are similar. The donor membrane is primed by asmall GTPase, which binds to the membrane in its GTP bound state, resulting in recruitment of apolymeric coat that causes the membrane to invaginate and form a vesicle (Schekman and Orci,1996). In some cases additional proteins are required for final fission of the vesicle from the donormembrane. Once the vesicle has detached from the membrane, GTP or ATP hydrolysis results inthe dissociation of the coat. Docking and fusion at the target membrane is the result ofinteractions between a number of proteins associated with both the vesicle and target membranes(Pelham, 2001). Specialised integral membrane proteins bring the membranes into close proximityand provide the force for membrane fusion. Three classes of vesicles have been well characterised.The proteins and mechanisms involved in the formation and fusion of these vesicles are discussedbelow. In addition to coated vesicles, as mentioned earlier large tubulovesicular carriers haverecently been described that are involved in long-range transport from the TGN to the plasmamembrane and from the ER to the Golgi (Stephens and Pepperkok, 2001).

4.1. Coat proteins and vesicle biogenesis

To maintain organelle integrity with the constant trafficking of proteins and lipids it isimportant that anterograde transport is selective and/or that it is balanced by selective recycling ofproteins and lipids in retrograde transport vesicles. Selective packaging of cargo can be throughactive concentration at exit sites, through interactions with cargo receptors or possibly throughinteractions with other cargo molecules (Herrmann et al., 1999). Many cargo molecules transitthrough more than one organelle while vesicle coats only bind to specific organelles. Thus, while itappears that cargo molecules are involved in coat recruitment, it is likely that other factors, suchas the membrane micro-environment, also play a role.

Three well-defined vesicle coats include the COPI and COPII and clathrin coats. Each coatfunctions in a specific pathway or set of pathways and results in transport of cargo from onemembrane compartment to another.

4.1.1. COPI

COPI coated vesicles mediate both intra-Golgi transport and retrograde transport from theGolgi to the ER. COPI vesicles were first identified as non-clathrin coated vesicles formed frompurified Golgi membranes when incubated with GTPgS and cytosol (Malhotra et al., 1989).Purification of the coat proteins revealed a complex of seven subunits, referred to as COPI orcoatomer (Malhotra et al., 1989; Waters et al., 1991).


COPI vesicle budding is initiated when the guanine nucleotide exchange factor (GEF) catalysesthe exchange of GDP for GTP in ARF1 (ADP-ribosylation factor 1) (Chardin et al., 1996).Membrane bound ARF-GTP recruits pre-assembled coatomer complexes, which cause themembranes to deform and form vesicles (Orci et al., 1993; Zhao et al., 1997). Coatomer, ARF1,and GTP are the minimum requirements for the formation of COPI vesicles from syntheticliposomes containing acidic phospholipids (Spang et al., 1998). However, the generation of COPIvesicles from liposomes with lipid compositions resembling Golgi membranes required anadditional component, namely the cytoplasmic tail of an abundant transmembrane protein p24(Bremser et al., 1999).

The p24 family of proteins were the first transmembrane proteins identified in COPI-coatedvesicles (Stamnes et al., 1995). The cytoplasmic domain of p24 interacts with COPI, promotingmembrane association of the coat complex and vesicle formation (Bremser et al., 1999). Thecytoplasmic tails of p24molecules contain the KKXX sequence that acts to recruit COPI tomembranes (Fiedler et al., 1996). It was initially proposed that p24 proteins could act as cargoreceptors, binding soluble cargo with their lumenal domains, and linking these to COPI coats(Fiedler et al., 1996). However, it now appears that the function of p24 family members may be toselect cargo by an indirect mechanism by excluding resident proteins, rather than through directinteractions with cargo molecules (Kaiser, 2000; Springer et al., 2000).

The COPI coat dissociates following GTP hydrolysis by ARF1. This GTP hydrolysis isactivated by a GTPase activating protein, known as ARF GAP (Cukierman et al., 1995).Interactions between coatomer and ARF1 dramatically increase the rate of GTP hydrolysis(Goldberg, 1999).

The ARF1 GEF is sensitive to the fungal metabolite brefeldin A, resulting in inactivation ofARF1 and cessation of COPI vesicle biogenesis (Donaldson et al., 1990; Chardin andMcCormick, 1999). Cells treated with brefeldin A or with mutations in one of the COPI genesshow defects in protein secretion, findings which were initially interpreted to indicate that COPIvesicles were involved in anterograde protein traffic (Pepperkok et al., 1993). However,cumulative evidence as discussed earlier now strongly suggests that COPI vesicles function inretrograde transport. The KKXX sequence is necessary and sufficient to direct retrogradetransport of membrane proteins from the Golgi to the ER (Jackson et al., 1993). The KKXXsequence is found in proteins such as the KDEL receptor, which binds to lumenal ER proteinscontaining the KDEL retrieval motif and returns them to the ER (Lewis and Pelham, 1992). Twodistinct approaches demonstrated that COPI coat proteins were able to recognise the KKXXsignal. Firstly, biochemical studies showed that coatomer binds to the KKXX motif at thecarboxy-terminus of resident ER membrane proteins (Cosson and Letourneur, 1994). Secondly,many of the yeast mutants that fail to retain a KKXX-tagged reporter molecule in the ER havemutations in the gene (RET1) which encodes the yeast a-COP protein (Letourneur et al., 1994).Thus, it is likely that the secretion defects observed in COPI mutants and following brefeldin Atreatment are a secondary effect, resulting from the failure to recycle components necessary forforward transport.

4.1.2. COPIIThe existence of a coat associated with anterograde ER to Golgi traffic, called COPII, was

demonstrated by the use of yeast mutants with defects in secretion (Kaiser and Schekman, 1990).


Genetic screens of these mutants revealed four genes that were essential for transport of a-factorprecursor from the ER, namely SEC12, SEC13, SEC16, SEC23 (Kaiser and Schekman, 1990).The products of these genes are involved in the biogenesis of COPII coated vesicles from ERmembranes.

The COPII coat is comprised of two heterodimeric complexes, Sec23p/24p and Sec13p/31p(Barlowe et al., 1994). The small GTPase, Sar1p, is activated by Sec12p, an ER integral membraneprotein that catalyses the exchange of GDP for GTP (Barlowe and Schekman, 1993). Sar1p-GTPbinds to ER membranes and recruits Sec23p/24p, which in turn recruits Sec13p/31p (Schekmanand Orci, 1996). Polymerisation of these subunits deforms the membrane and causes vesicleformation (Barlowe et al., 1994). In a manner similar to COPI coats, GTP-hydrolysis results indissociation of the COPII coat. Sec23p acts as the GTPase activating protein (GAP) for Sar1p(Yoshihisa et al., 1993). The GAP activity of Sec23p is increased 10-fold in the presence of Sec13p/31p (Antonny et al., 2001).

The requirements for generation of COPII vesicles from ER membranes in vitro are Sar1p,Sec23p/24p and Sec13p/31p (Barlowe et al., 1994). Three additional proteins- Sec7p, Sec16p andYpt1p-associate with COPII vesicles when crude cytosol is used in the budding reaction(Schekman and Orci, 1996). The vesicles formed in these in vitro assays can fuse with Golgimembranes but not ER membranes, indicating that they are involved in anterograde transportfrom the ER to the Golgi (Rexach et al., 1994).

It has been suggested that COPII vesicle formation may be linked to cargo recruitment (Kuehnand Schekman, 1997). However, the proposal of a di-acidic (DXE) export signal in the cytosolicportion of several integral membrane proteins (Nishimura and Balch, 1997) was subsequentlyshown to be incorrect and further studies revealed that this di-acidic signal is important for cargoconcentration downstream of COPII budding (Nishimura et al., 1999). Also it has beendemonstrated that COPII coats can form in the absence of cargo in the ER (Yeung et al., 1995).Thus, vesicle budding and cargo selection in many cases are likely to be separate processes.Nonetheless, several cargo proteins appear to be concentrated in COPII vesicles leaving the ER,suggesting that there may be cargo receptors within these vesicles that also interact with the coat(Herrmann et al., 1999). One such receptor, Erv29p, has recently been shown to interact directlywith glycosylated pro-a-factor and is necessary for packaging of this soluble cargo protein intoCOPII vesicles (Belden and Barlowe, 2001). It is possible that SNARE molecules act as receptorsfor COPII vesicle formation as the SNAREs Bet1p and Bos1p interact directly with Sar1p andSec23p/24p (Springer and Schekman, 1998).

4.1.3. Clathrin

The first membrane coat to be identified was the clathrin coat (Kanaseki and Kadota, 1969;Schmid, 1997). Clathrin coated vesicles are involved in transport of proteins from the TGN toendosomes and from the plasma membrane to endosomes. Clathrin light and heavy chainsassemble into triskelions structures that self polymerise to form a polygonal lattice, or cage-likestructure, resulting in curvature of the membrane (Marsh and McMahon, 1999). Clathrintypically associates with membranes via a heterotetrameric complex called the adaptor proteincomplex (AP) (Schmid, 1997). The four subunits of AP are referred to as adaptins. There aremultiple adaptins that combine to form several classes of AP. Each AP consists of two large, amedium and a small subunit, forming a structure likened to ‘‘Mickey Mouse’’ (Robinson and


Bonifacino, 2001). The ‘‘head’’ consists of the amino-terminal domains of the two large subunits,together with the small and medium subunits, whereas the ‘‘ears’’ are the carboxy-terminaldomains of the two large subunits (Robinson and Bonifacino, 2001).

APs interact with both clathrin and membrane cargo acting as a bridge linking clathrin tomembranes. Different APs associate with different membranes through interactions with specificcargo (Schmid, 1997). Clustered cargo with attached adaptors recruit clathrin which provides thescaffold for the distortion and invagination of the membrane creating a clathrin-coated pit(Marsh and McMahon, 1999). Fission of the vesicle from the donor membrane requires theactivity of the GTPase dynamin (van der Bliek et al., 1993). Dynamin self-assembles into a collarof helices around the neck of the invaginated membrane, and GTP hydrolysis allows dynamin toact as a ‘‘pinchase’’ to sever the coated vesicle from the membrane (Hinshaw and Schmid, 1995;Sweitzer and Hinshaw, 1998). Following budding, auxillin binds to the clathrin coat and recruitsthe chaperone heat shock cognate 70 (Hsc70) which catalyses the disassembly of the clathrin coatcoupled to ATP hydrolysis (Ungewickell et al., 1995).

4.1.4. Adaptor protein complexesTo date, four adaptor protein complexes have been identified. AP1 is associated with TGN and

endosomal membranes. Binding of AP1 to membranes requires activation of the GTPase ADPribosylation factor 1 (ARF1). AP1 interacts with the cytoplasmic tail of M6P receptor (Le Borgneet al., 1996) and has a role in transport of Man-6-P-tagged lysosomal enzymes to lysosomes viaendosomes (Le Borgne and Hoflack, 1998). It was initially thought that AP1 was involved inTGN to endosome transport of these enzymes but it now appears that it is involved in retrogradetransport of the receptors back to the TGN for successive rounds of transport (Robinson andBonifacino, 2001). Support for this model comes from the observation that cells from micedeficient in AP1 accumulate M6P receptors in the endosomes as the receptors fail to recycle backfrom the endosomes to the TGN (Meyer et al., 2000).

AP2 vesicles function in the transport of plasma membrane receptors to endosomes (Schmid,1997). AP2 associates with the plasma membrane through interactions with specific phosphoi-nositides at the plasma membrane (Kirchhausen, 1999; Ford et al., 2001) and by binding to theinternalisation signals of various plasma membrane receptors. These signals include the sequenceYXXF (where X is any amino acid and F is a bulky hydrophobic amino acid) found in severalproteins, including the transferrin receptor (Bonifacino and Dell’Angelica, 1999).

AP3 is localised to the TGN and endosomes. AP3 is involved in targeting specific proteins tolysosomes or related compartments, such as melanosomes (Jackson, 1998). Significantly,mutations in AP3 subunits in yeast, Drosophila, mice and humans cause lysosomal storagedefects (Rohn et al., 2000). AP3 also appears to play a role in synaptic vesicle biogenesis as miceand flies with these mutations have neurological defects (Rohn et al., 2000).

Another TGN-localised AP, AP4, has been identified in some organisms and appears to beinvolved in TGN to endosome trafficking but so far has not been shown to associate with clathrin(Robinson and Bonifacino, 2001).

4.1.5. Adaptor-related proteinsRecently a number of groups have discovered a new family of adaptor-related proteins (Boman

et al., 2000; Dell’Angelica et al., 2000; Hirst et al., 2000; Poussu et al., 2000). Called GGAs for


Golgi-localised, c ear containing, ARF-binding proteins, they were identified either due to theirhomology to the ‘‘ear’’ domain of the AP subunit g-adaptin (Dell’Angelica et al., 2000; Hirst et al.,2000), or because of their ability to bind activated-ARF (Boman et al., 2000).

In contrast to the heterotrimeric adaptor complexes, GGAs are monomeric proteins with aunique four domain structure (Boman, 2001; Robinson and Bonifacino, 2001). The N-terminalVHS (Vps27p, Hrs, STAM) domain binds to the cytoplasmic tails of three proteins that trafficfrom the TGN to endosomes: sortilin (Nielsen et al., 2001), the cation-independent M6P receptor(Puertollano et al., 2001a) and cation-dependent M6P receptor (Zhu et al., 2001). The domaincontiguous with the VHS domain is the GAT domain, so-called as homology is shared amongstGGA and Tom1 proteins. The GAT domain binds ARF-GTP, resulting in a stabilised membraneassociated complex (Puertollano et al., 2001b). The GAT domain is both necessary and sufficientfor Golgi localisation (Dell’Angelica et al., 2000). A hinge domain connects the GAT and the C-terminal GAE (c-adaptin ear) domains and, together with the GAE domain, binds clathrinmolecules, recruiting them to budding vesicles (Puertollano et al., 2001b). It now appears likelythat GGAs act to transport M6P receptors to endosomes, while AP1 acts to recycle thesereceptors back to the TGN.

4.2. Vesicle docking and fusion

While selective packaging of cargo into vesicles is essential for the maintenance of organelleintegrity, so too is the appropriate targeting and fusion of vesicles with acceptor membranes.Vesicle docking and fusion occurs in three steps. First, the vesicle is tethered to the targetmembrane by tethering complexes. Second, if the target membrane and vesicle membrane havecompatible fusion markers (SNAREs), they will interact, docking the vesicle onto the targetmembrane. Third, these fusion markers form a tight complex, bringing the membranes closetogether and promoting membrane fusion. Following fusion, the fusion markers from the vesiclemust be recycled to the donor membrane for further fusion events.

4.2.1. SNAREsAt the heart of the basic fusion machinery is a family of integral membrane proteins present on

both the vesicle and target membranes, called SNAREs (soluble NSF attachment proteinreceptors) (S .ollner et al., 1993b). These fusion markers play a role in both the recognition ofcompatible membranes and in membrane fusion (Weber et al., 1998). SNAREs were identifiedthrough both biochemical and genetic analysis of components involved in membrane fusion(Novick et al., 1981; Nichols and Pelham, 1998). The name SNARE comes from the interactionwith two components that were identified through in vitro transport assays. One component is anATPase that is sensitive to N-ethylmaleimide (NEM) named NEM-sensitive factor (NSF) (Blocket al., 1988). The other, an interacting protein called soluble NSF attachment protein or SNAP(Clary et al., 1990).

The cytoplasmic coiled coil domains of SNAREs interact to form very stable four-helixbundles, referred to as SNARE complexes or SNARE-pins (Weber et al., 1998). The formation ofa SNARE complex brings the membranes into close proximity allowing membrane fusion tooccur, either directly via SNARE associations (Weber et al., 1998; McNew et al., 2000b) orthrough downstream effectors (Ungermann et al., 1998). SNAREs have distinct cellular


localisations with interacting SNAREs present on both the vesicle (v-SNARE) and target (t-SNARE) membranes. When SNARE complexes form between two membranes they are referredto as trans-SNARE pairs. Following fusion they are called cis-SNARE pairs as the interactingSNAREs are present on the same membrane. NSF and SNAP act together to disrupt cis-SNAREcomplexes. NSF binds to assembled SNARE complexes via SNAP and catalyses ATP-hydrolysis,resulting in disassembly of the SNARE complex (S .ollner et al., 1993a; Mayer et al., 1996). v-SNAREs are then transported back to the donor membrane for further rounds of transport.Inappropriate trans-SNARE assembly is inhibited by the Sec1 family of proteins (Aalto et al.,1992). Sec1-like proteins bind to t-SNAREs and regulate SNARE complex formation (Pevsneret al., 1994; Bryant and James, 2001).

The fact that SNAREs have discrete localisations at either the vesicle or target membrane andcan interact specifically to form SNARE complexes, led to the SNARE hypothesis, whichpredicted that membrane fusion specificity was provided by SNARE molecules (S .ollner et al.,1993b; Rothman, 1994). Several findings, however, questioned whether a bi-molecular v-SNARE/t-SNARE interaction was alone sufficient to specify membrane targeting. First, neuronal t-SNAREs are distributed throughout the plasma membrane although exocytosis occurs only atspecific sites in the presynaptic membrane (Garcia et al., 1995). Second, a single v-SNARE isinvolved in more than one transport step in vivo (Lupashin et al., 1997; von Mollard et al., 1997).Third, soluble SNAREs have been shown to interact promiscuously in vitro (Tsui and Banfield,2000).

Recent studies by Rothman and S .ollner and colleagues using purified SNAREs integrated intoliposomes have indeed suggested a high level of specificity in v-SNARE-t-SNARE interactions(McNew et al., 2000a; Parlati et al., 2000). These assays are likely to represent a more realisticsituation than the assays showing SNARE promiscuity as the SNAREs are loaded ontoliposomes, rather than in solution. These liposome experiments also demonstrated a strictrequirement for three SNAREs in one membrane and one in the other, to generate a specific four-helix bundle and a functional complex (Parlati et al., 2000). These findings provide a deeperunderstanding of the specificity of membrane fusion events, and explain how any single SNAREmolecule can be involved in multiple transport steps, as different combination of SNAREs displaydifferent fine specificities.

However, while SNAREs clearly impart a level of specificity on vesicle fusion, there must beadditional levels of regulation. This is in part to account for the fact that SNAREs cycle betweenvesicle and target membranes and are therefore present on several membranes and yet fusion ishighly specific. Additionally, vesicles can still attach to membranes when SNAREs are cleaved byneurotoxins, suggesting that SNAREs are not required for initial recognition (Broadie et al.,1995). Thus, it is likely that there are several sets of interactions responsible for vesicle targeting.A number of proteins have been identified that function prior to SNARE interactions andmembrane fusion. These include the small GTPases, Rabs, and their effector molecules (Watersand Hughson, 2000; Segev, 2001).

4.2.2. Rabs

Rabs are key regulators of vesicle docking and fusion. These small Ras like GTPases actupstream of SNARE proteins in the initial tethering of vesicles to target membranes. Rabs recruiteffector proteins to one or both membranes. These effectors are involved in the initial attachment


of vesicles to membranes, allowing SNAREs to interact (Pfeffer, 1999; Waters and Pfeffer, 1999).Rabs have a very restricted localisation with each member being associated with a particularorganelle or transport step (Rodman and Wandinger-Ness, 2000).

Rab function is regulated at many levels. Rabs cycle between the cytosol and membrane andcan be switched on and off by proteins regulating the GTP/GDP-bound state of the protein(Chavrier and Goud, 1999). Regulators of Rab function include guanine nucleotide exchangefactors (GEFs) that catalyse GDP-GTP exchange and GTPase activating proteins (GAPs) thatstimulate the low intrinsic GTPase activity of Rabs (Segev, 2001). Two additional proteins controlRab function by regulating the membrane attachment of Rab-GDP to membranes. GDP-dissociation inhibitor (GDI) extracts Rab-GDP from the membrane and maintains it in itsinactive form, while GDI-displacement factor (GDF), displaces GDI and recruits Rab tomembranes (Gonzalez and Scheller, 1999). The proteins that regulate Rab activation receivesignals from upstream effectors thereby linking transport events to general cellular processes aswell as to external events (Segev, 2001). Each Rab protein has its own specific set of regulatoryproteins (Gonzalez and Scheller, 1999).

Rabs have also been shown to have a role in vesicle transport through interactions with thecytoskeleton (Nielsen et al., 1999). Thus each Rab may regulate a single transport event atmultiple levels.

4.2.3. Tethers

The key effectors of Rab function in vesicle attachment are known as tethers (Pfeffer, 1999;Waters and Pfeffer, 1999). These tethering molecules are recruited to membranes by activated Rabproteins and form a bridge between the vesicle and the target membrane prior to SNARE-complex assembly and fusion. Unlike Rabs and SNAREs that form large families of relatedproteins that function at different transport steps, tethers that function in distinct pathways arenot related. Tethering molecules are generally either long coiled coil proteins or large multimericcomplexes (Pfeffer, 1999; Waters and Pfeffer, 1999).

Mutations in tethering complexes can be overcome by over-expression of the relevant Rab orSNARE molecules, indicating that tethers act upstream of SNARE complex assembly(Sapperstein et al., 1996). Tethers are not essential for vesicle transport but increase the efficiencyof vesicle docking and fusion by bringing the membranes together, promoting SNARE complexassembly (Cao et al., 1998).

Tethers have been identified for a number of transport steps. These include ER to Golgi traffic,where three different tethers have been identified. These are the long coiled coil molecule, Uso1p,and two large complexes, namely the Sec34p/Sec35p complex and TRAPP (Lowe, 2000). All threetethers interact genetically indicating that they function at the same transport step.

The mammalian homolog of Uso1p, p115, has been shown to function in intercisternal Golgitransport (Waters et al., 1992; Seemann et al., 2000), ER to Golgi traffic (Alvarez et al., 1999) andGolgi reassembly following mitosis (Rabouille et al., 1995b). p115 interacts with two other coiledcoil proteins: giantin on COPI coated vesicles (Sonnichsen et al., 1998) and GM130 on Golgimembranes (Nakamura et al., 1997). It is likely that these three molecules form a long tetherjoining the vesicle to the Golgi membrane, however a complex containing all three proteins is yetto be isolated. Long fibrous tethers have been observed linking vesicles to Golgi membranes in


electron micrographs (Orci et al., 1998). Immuno-labelling of these preparations may establish ifthese proteins are present in these Golgi associated tethers.

p115 is also loaded onto COPII vesicles leaving the ER in a Rab1 dependent manner (Allanet al., 2000). Recent experiments suggest that Rab1 coordinates transport from the ER to Golgithrough interactions with different tethers at the donor and acceptor membranes (Moyer et al.,2001).

Another tethering protein that has been the focus of intensive studies is the early endosomeantigen 1 (EEA1) (Mu et al., 1995). EEA1 forms a long coiled coil dimer with two FYVE fingerdomains and two Rab5 binding domains at each end (Callaghan et al., 1999). EEA1 is involved inearly endosome docking and fusion (Mills et al., 1998; Simonsen et al., 1998; Christoforidis et al.,1999a). EEA1 associates with early endosomal membranes via its Rab5 binding domains and itsFYVE finger domains that bind to phosphatidylinositol-3-phosphate (PI(3)P) in the membrane(Stenmark et al., 1996; Burd and Emr, 1998; Christoforidis et al., 1999a; Kutateladze et al., 1999).Rab5 promotes the localised generation of PI(3)P by interacting with the phosphatidylinositol-3-OH kinase (Christoforidis et al., 1999b). Membrane bound EEA1 forms part of a large complexthat includes Rabaptin5-Rabex5, NSF and the t-SNARE syntaxin 13 (McBride et al., 1999).EEA1 also interacts with syntaxin 6, a SNARE implicated in TGN to early endosome transport(Simonsen et al., 1999). The multiple protein–protein interactions of EEA1 suggest that it mayfunction as a scaffolding protein bringing the various proteins necessary for fusion into closeproximity, or that it has a coordinating role in regulating membrane fusion.

Other tethers that have been characterised include the exocyst (or Sec6/Sec8 complex), which isinvolved in Golgi to plasma membrane transport (Hsu et al., 1996; TerBush et al., 1996; Yeamanet al., 2001), and the Vam2/6p complex involved in vacuole fusion in yeast (Price et al., 2000;Wurmser et al., 2000).

5. Localisation signals in the secretory pathway

Each organelle of the secretory pathway allows the transit of a multitude of proteins whilemaintaining its own unique set of resident proteins that define its structure and function. Thelocalisation of proteins to specific organelles within eukaryotic cells depends on discrete targetingsignals contained within these (glyco)proteins. The targeting signals for a number of residentproteins have been identified, however, in many cases the underlying mechanisms responsible forthe recognition of these signals remain poorly defined.

5.1. ER retention and retrieval

Localisation of ER proteins is generally achieved through two complementary mechanisms:retention and retrieval (Teasdale and Jackson, 1996). Particular sequences within ER proteinsfunction to retain the protein in the ER, while additional sequences are required to retrieveescaped ER proteins from downstream compartments.

Soluble ER proteins contain the carboxy-terminal tetrapeptide, -KDEL or -HDEL, which isnecessary for their retrieval from the Golgi (Munro and Pelham, 1987; Pelham et al., 1988). TheH/KDEL sequence is recognised by the Golgi localised H/KDEL receptor, Erd2 (Lewis and


Pelham, 1990; Semenza et al., 1990). The Erd2 receptor binds the H/KDEL sequences in theslightly acidic environment of the Golgi and releases them upon arrival at the ER (Wilson et al.,1993). Deletion of H/KDEL sequences from ER proteins results in their secretion, albeit slowly,while addition of H/KDEL to secreted proteins results in redistribution of these proteins to theER (Munro and Pelham, 1987). However, removal of the HDEL sequence from an essential ERprotein, Kar2p (BiP), was found to have no effect on cell viability (Semenza et al., 1990),suggesting that sufficient levels of Kar2p were being retained in the ER, and implying additionalmechanism(s) for ER localisation.

Studies of several ER proteins have indeed revealed a dual mechanism for ER localisation. ACa2+-dependent retention mechanism has been identified as the dominant signal for steady-stateER localisation of the soluble ER protein, calreticulin (S .onnichsen et al., 1994). On the otherhand, the KDEL sequence at the C-terminus of calreticulin was necessary for retrieval of smallamounts of calreticulin that had escaped to the Golgi (S .onnichsen et al., 1994). Thus, retention isthe predominant mechanism for maintaining calreticulin in the ER, whereas retrieval plays a rolein increasing the efficiency of localisation.

The KDEL receptor (Erd2p) is packaged into COPI-coated vesicles at the Golgi. Like other ERtype I transmembrane proteins, Erd2p contains a di-lysine retrieval (Lewis and Pelham, 1990;Semenza et al., 1990). The –K(X)KXX consensus sequence interacts with the COPI coat (Cossonand Letourneur, 1994; Letourneur et al., 1994). Thus, Erd2p binds KDEL tagged proteins andreturns them to the ER in COPI vesicles. In addition to its role as a retrieval signal, the di-lysinemotif in other membrane proteins may also act as a retention signal. Chimeras containing KKAAtags are actively retained in the ER and this localisation is not affected in COPI mutant cell lines(Andersson et al., 1999).

Type II transmembrane proteins have a di-arginine motif for ER localisation with two arginineslocated within the +2 to +5 positions of the amino acid sequence, either side by side or separatedby one amino acid (Schutze et al., 1994). Addition of this motif to a cell surface type II proteinresults in efficient targeting to the ER (Schutze et al., 1994).

Yeast Sec12p is a type II transmembrane protein that is essential for the formation of ER-derived transport vesicles (Schekman and Orci, 1996). Under steady state conditions Sec12p islocated in the ER, however, the majority of the Sec12p molecules have been processed by a cis-Golgi specific enzyme, which adds an a-1-6 mannose residue, indicating that Sec12p cyclesthrough the Golgi (Nakano et al., 1988). Optimal ER localisation of Sec12p is dependent on aGolgi localised protein, Rer1p (Nishikawa and Nakano, 1993; Sato et al., 1995). Rer1p is likely tobe a receptor to recycle Sec12p molecules that have escaped to the Golgi. Sato and colleagues havealso identified a retention mechanism for the ER localisation of Sec12p that is distinct from theRer1p-dependent retrieval mechanism (Sato et al., 1996). Whereas the transmembrane domain ofSec12p is necessary for retrieval, the cytoplasmic domain is involved in retention (Sato et al.,1996). Thus, it appears that both active retention and retrieval are necessary for optimallocalisation of ER proteins.

5.2. Golgi localisation

A striking feature of the Golgi apparatus is its ability to maintain a resident set of proteinsagainst the flow of secretory cargo. A number of targeting signals have been identified for resident


Golgi proteins, nonetheless, despite intensive research, the mechanisms of localisation remain byand large elusive. Considering the array of different classes of resident Golgi proteins, it is likelythat there is a diversity of signals and mechanisms to account for their localisation.

As for ER proteins, it is probable that both retention and retrieval play a role in maintenance ofGolgi localised proteins. A number of type I transmembrane proteins which are concentrated inthe TGN, cycle between the TGN and the plasma membrane (Luzio et al., 1990; Molloy et al.,1994). Sequences within the cytoplasmic tails of these proteins have been shown to be necessaryfor their retrieval from the plasma membrane. For example, TGN38 contains a tyrosine-basedmotif (SDYQRL) that is necessary for internalisation from the plasma membrane and transportto the TGN (Bos et al., 1993; Humphrey et al., 1993; Wong and Hong, 1993). This sequenceinteracts with the m2 subunit of AP2 (Ohno et al., 1995; Stephens et al., 1997) and is necessary forinternalisation in clathrin coated vesicles (Roquemore and Banting, 1998). The transmembranedomain of TGN38 can also act as a Golgi localisation signal and may function in Golgi retention(Ponnambalam et al., 1994).

The endoprotease furin has two separate signals within its cytoplasmic tail: one for retrievalfrom the plasma membrane and one for retention at the Golgi (Takahashi et al., 1995). Atyrosine-based motif (YKGL) is necessary for endocytosis, while an acidic cluster containing twoserine residues is necessary for TGN localisation (Jones et al., 1995; Takahashi et al., 1995).Phosphorylation of these serine residues by casein kinase II results in recruitment of PACS-1(phosphofurin acidic cluster-sorting protein), which connects furin with AP1 for transport fromendosomes to the TGN (Jones et al., 1995; Dittie et al., 1997; Wan et al., 1998). Thus, multiplesignals are involved in steady-state localisation of type I membrane proteins to the TGN.

The Menkes P type ATPase copper transporter is a multi-membrane spanning protein thatcycles between the plasma membrane and the TGN (Petris et al., 1996). Steady-state localisationof this protein is regulated by copper concentration, with high levels of copper shifting the proteinfrom the TGN to the plasma membrane (Petris et al., 1996). Again, both retention and retrievalare necessary for Golgi localisation. The third transmembrane domain of the Menkes protein actsas a TGN localisation signal and can target a reporter protein to the Golgi (Francis et al., 1998),while a di-leucine motif near the C-terminus of the protein is necessary for steady-statelocalisation (Petris et al., 1998). This di-leucine motif serves as an internalisation signal as it isnecessary for retrieval of the protein from the plasma membrane (Petris et al., 1998; Francis et al.,1999).

5.2.1. GlycosyltransferasesThe most intensively studied integral membrane proteins of the Golgi are the glycosyltrans-

ferases. These type II membrane proteins show distinct but overlapping distributions across theGolgi stack (Rabouille et al., 1995a). For over a decade many studies have attempted to identifysignals in glycosyltransferases that are responsible for their segregation from secretory traffic, andlocalisation within discrete sections of the Golgi apparatus. Collectively, these studies have beenunable to identify specific targeting sequences, but have nonetheless identified regions of theglycosyltransferases that are critical for their compartment-specific localisation (Colley, 1997;Gleeson, 1998; Munro, 1998).

All Golgi glycosyltransferases share a common topology and domain structure, consisting of ashort amino-terminal cytoplasmic tail, a transmembrane domain, and a lumenal domain which


includes the catalytic domain and a ‘stem’ between the membrane and catalytic domain (Paulsonand Colley, 1989). Despite this common domain structure, there is very little sequence homologybetween the different glycosyltransferases.

Most studies that have identified targeting signals of glycosyltransferases have used deletionmutants and fusions with non-Golgi proteins. A consistent finding is that the transmembranedomain of many different glycosyltransferases provides a dominant localisation signal. A numberof studies have also shown contributions from the lumenal domain and cytoplasmic tail,suggesting that there may be multiple signals involved in the specific localisation of these Golgienzymes (Colley, 1997; Gleeson, 1998; Milland et al., 2002).

A difficulty in defining bone fide Golgi targeting signals of glycosyltransferases is the potentialof the glycosyltransferase chimeric molecules to form complexes with other endogenous Golgimembrane components. This raises the possibility that Golgi targeting information may bepresent in other components of the complex. Thus, the Golgi localisation of some of theglycosyltransferase chimeras may be due solely to sequences that promote their interaction withexisting membrane complexes. This could be especially pertinent to the ability of the lumenaldomain of some of the glycosyltransferases to target chimeras to the Golgi. For example, a solubleform of GlcNAc-TI, containing only the lumenal domain, but lacking the transmembrane domainthat has previously been shown to function as a localisation signal (Burke et al., 1992; Tang et al.,1992), accumulates in the Golgi prior to secretion (Opat et al., 2000). GlcNAc-TI has been shownto exist as high molecular weight complexes and analysis of membrane-bound GlcNAc-TIchimeras indicates that the formation of these complexes does not require the transmembranedomain or cytoplasmic sequences of GlcNAc-TI (Opat et al., 2000). Thus, it is likely that inclusioninto high molecular weight complexes occurs through interactions with the lumenal domain, andcan account for the Golgi localisation of both the membrane-bound GlcNAc-TI chimera andsoluble GlcNAc-TI.

Based on the observation that the transmembrane domain of glycosyltransferases contains adominant localisation signal, two models were initially proposed to explain the localisation ofresident enzymes to the Golgi apparatus. One model is based on lipid bilayer sorting and the otheron oligomerisation of Golgi resident proteins.

Lipid bilayer sorting: The lipid bilayer sorting model, proposed by Bretscher and Munro (1993)and Masibay et al. (1993) is centred around four observations. First, mutational analysis of thetransmembrane domains of Golgi enzymes failed to identify individual residues or sequencemotifs involved in localisation indicating that a more general property of the hydrophobic regionis relevant (Munro, 1991; Masibay et al., 1993). Second, lipid bilayer thickness increases along thesecretory pathway (Nezil and Bloom, 1992) due to an increase in cholesterol and sphingolipidconcentration from ER membranes to the plasma membrane (Orci et al., 1981; Moreau andCassagne, 1994). Third, Golgi localised proteins tend to have shorter transmembrane domainsthan plasma membrane proteins (Munro, 1995a). Fourth, a recent computational comparisonbetween Golgi residents and proteins which move through the Golgi showed that the overallhydrophobicity of the transmembrane domains of Golgi resident proteins was significantly lower(Yuan and Teasdale, 2002). Munro suggested that the thinner bilayer of the Golgi maypreferentially retain resident enzymes which generally have shorter transmembrane domains. Forexample, if the transmembrane domain of a2; 6-sialyltransferase is increased from 17 to 23 aminoacids, a2; 6-sialyltransferase is found on the plasma membrane (Munro, 1991). While, shortening


the transmembrane domain of a plasma membrane protein from 23 to 17 residues reducestrafficking to the cell surface, resulting in accumulation in the Golgi apparatus (Munro, 1995b).

An increasing number of studies have found that the transmembrane domain does not fullyaccount for the localisation of Golgi enzymes (Burke et al., 1994; Graham and Krasnov, 1995; Maet al., 1997; Chen et al., 2000; Opat et al., 2000; Milland et al., 2002). Studies of GlcNAc-TIchimeras expressed at modest levels in stable cell lines have demonstrated that efficientlocalisation of GlcNAc-TI requires more than one domain, however chimeras containing only onedomain, either the transmembrane or lumenal domain, were also retained in the Golgi, albeit atlower levels (Burke et al., 1994). Colley and colleagues have identified an isoform of a2; 6-sialyltransferase that requires lumenal sequences in addition to the transmembrane domain forGolgi localisation (Ma et al., 1997; Chen et al., 2000).

Oligomerisation: The aggregation or oligomerisation model was first proposed by Machamer(1991), who suggested that glycosyltransferases are induced to form aggregates when they reachthe correct Golgi compartment. At the time, Machamer proposed that the interaction between thetransmembrane domains of glycosyltransferase and lipid bilayers of Golgi membranes may be thedriving force for oligomerisation. An extension of the aggregation model was put forward byNilsson who suggested that different enzymes from the same Golgi compartment interact witheach other (kin recognition) forming large hetero-oligomeric structures (Nilsson et al., 1993).These aggregates would form upon arrival at the correct cisternal environment and be excludedfrom forward moving vesicles. Nilsson et al. (1994) demonstrated that two medial-Golgi enzymesinteract with each other by the addition of an ER retention signal to the GlcNAc-TI cytoplasmictail that not only caused GlcNAc-TI to localise to the ER but also partially retained anothermedial-Golgi enzyme, Man II, within the ER. In contrast the trans-Golgi enzyme, b1,4-galactosyltransferase, was not relocated to the ER in these experiments, indicating specificassociation of enzymes from the same compartment. Although this ‘kin recognition’ interactionwas initially suggested to occur via the transmembrane domain it was subsequently shown that thestem region of GlcNAc-TI, and not the transmembrane domain, was responsible for associationwith Man II (Nilsson et al., 1996).

A number of more recent studies have identified glycosyltransferases in high molecular weightcomplexes (Jungmann and Munro, 1998; Chen et al., 2000; Opat et al., 2000; Sasai et al., 2001).The formation of these complexes was found to increase the efficiency and stability of Golgilocalisation. The lumenal domains of the medial-Golgi enzymes GlcNAc-TI, GlcNAc-TII andGlcNAc-TV are required for the formation of high molecular weight complexes and retention inthe Golgi (Opat et al., 2000; Sasai et al., 2001). For the late Golgi enzyme a2; 6-sialyltransferaseoligomers only formed at pH 6.3, corresponding to the low pH of the late Golgi (Chen et al., 2000).

There is increasing evidence for the existence of multi-enzyme complexes within the Golgi. Theyeast enzyme Mnn9p co-precipitates with a number of proteins which bear homology toglycosyltransferases and activity assays on the Mnn9p immunoprecipitates indicated the presenceof multiple mannosyltransferase activities (Jungmann et al., 1999). Recent studies on sphingolipidsynthesis, employing both in vitro biochemical approaches and in vivo fluorescence resonanceenergy transfer (FRET) analysis, revealed that two glycosyltransferases involved in sequentialsteps of glycolipid synthesis are physically associated (Giraudo et al., 2001).

The cisternal maturation model of Golgi transport predicts that intra-Golgi retrogradetransport of glycosyltransferases would represent a major mechanism for their localisation. Using


in vitro assays, medial-Golgi resident enzymes have been found to be concentrated into COPIvesicles (Lanoix et al., 1999, 2001). Further, transport intermediates containing resident Golgienzymes have been shown to fuse preferentially with the cis-Golgi compartment (Love et al.,1998). Although at steady state GlcNAc-TI is concentrated within the medial-Golgi, newlysynthesised GlcNAc-TI has been shown to be transported rapidly through the Golgi stack to thetrans-Golgi network (Opat et al., 2001). Therefore, specific features of retrograde transport fromthe TGN are likely to be important in establishing the gradient of GlcNAc-TI activity throughoutthe Golgi stack. Although the above findings highlight the potential importance of retrogradetransport pathways in establishing the asymmetric distribution of Golgi glycosyltransferasesacross the stack, our understanding of intra-Golgi retrograde recycling remains rudimentary.

In summary, it is clear that there is no discrete signal involved in Golgi localisation ofglycosyltransferases. Instead, localisation may involve disparate regions of the molecule. Eitherlipid-mediated sorting or oligomerisation may mediate the retention of the enzymes within Golgicisternal membranes and/or regulate their inclusion into retrograde transport vesicles (Munro,1998).

5.2.2. Peripheral Golgi membrane proteins

A wide range of cytosolic proteins associate peripherally with Golgi membranes and have rolesin vesicle trafficking and maintenance of Golgi structure. Many of these proteins associate withspecific Golgi compartments. The Golgi targeting signals for a number of peripheral membraneproteins have been identified. The molecular mechanisms associated with the recruitment of thesetargeting determinants have generated considerable current interest and appear to be oftenassociated with activated small G proteins and/or lipids (Munro, 2002).

Cytoplasmic coat proteins interact with membranes by binding to specific sequences in cargoproteins. For example, AP1 binds to the cytoplasmic tail of the MP6 receptor (Le Borgne et al.,1996), and COPI binds to KKXX motifs in the cytoplasmic tails of various ER membraneproteins (Teasdale and Jackson, 1996).

A complex of three proteins, namely giantin, GM130 and p115 has been shown to be involvedin tethering of COPI vesicles to Golgi membranes (Sonnichsen et al., 1998) and reassembly of theGolgi membrane stack following mitosis (Shorter and Warren, 1999; Dirac-Svejstrup et al., 2000).Giantin is a type II membrane protein (Linstedt and Hauri, 1993), while GM130 and p115 areboth peripheral membrane proteins (Waters et al., 1992; Nakamura et al., 1995). The extreme C-terminus of GM130 associates with cis-Golgi membranes by binding to the N-myristoylated Golgireassembly stacking protein GRASP65 (Barr et al., 1997, 1998). The C-terminal acidic domain ofp115 interacts with the N-terminus of GM130 (Nakamura et al., 1997; Nelson et al., 1998).Therefore, it is possible that the interaction between p115 and GRASP65/GM130 results in Golgilocalisation of p115. However, Sztul and colleagues have shown that mutants of p115 lacking theGM130-binding domain are targeted to the Golgi in transfected COS cells, and that the N-terminal head domain of p115 is required for Golgi localisation in vivo (Nelson et al., 1998). Thus,the head domain of p115 may be required for Golgi localisation, while the C-terminus functionsto link giantin to GM130 to facilitate vesicle docking and Golgi reassembly.

Oxysterol binding protein (OSBP) binds to Golgi membranes via its pleckstrin homology (PH)domain (Levine and Munro, 1998). This domain binds to phosphatidylinositol-4-phosphate(PI(4)P) on Golgi membranes in vivo, however, OSBP mutants that are unable to bind to PI4P are


still able to target to Golgi membranes, albeit less efficiently, in an ARF-dependent manner(Levine and Munro, 2002). Thus both PI(4)P binding and binding to another, as yet unidentified,ARF-regulated membrane component is necessary for optimal Golgi localisation of OSBP.

Recruitment of the vesicle biogenesis factor protein kinase D (PKD) to TGN membranesoccurs through an interaction of its C1a domain with diacylglycerol (DAG) (Baron andMalhotra, 2002). While PKD contains a PH domain, this domain is not necessary for recruitmentof this protein to the Golgi (Maeda et al., 2001). Instead, the PH domain is likely to recruitvarious effector proteins that are required for vesicle biogenesis to the budding site (Bankaitis,2002).

Recently, a family of Golgi coiled coil peripheral membrane proteins have been identified inanimal and yeast cells that contain a conserved B42 amino acid sequence at the C-terminus,called the GRIP domain (Barr, 1999; Kjer-Nielsen et al., 1999a; Munro and Nichols, 1999). GRIPproteins are considered to be important as either Golgi matrix proteins or as tethering moleculesin membrane transport. The level of sequence identity between the GRIP domains is only modest,however, functional assays have demonstrated that a number of different GRIP sequences canspecifically target reporter molecules to the Golgi apparatus. By analysis of chimeric greenfluorescent proteins (GFP), the GRIP domain has been shown to be both necessary and sufficientfor Golgi targeting in animal cells (Barr, 1999; Kjer-Nielsen et al., 1999a, 1999b; Munro andNichols, 1999). Furthermore, the GRIP domain of one family member, p230, has been shown totarget specifically to the TGN of mammalian cells (Brown et al., 2001). In addition, a coiled coilprotein with a GRIP sequence in protozoan parasites has recently been identified and the GRIPdomain shown to be specifically recruited to the TGN in the trypanosomatid parasite, Leishmaniamexicana (McConville et al., 2002). The identification of GRIP domain proteins associated withthe TGN of this very primitive protozoan eukaryotic cell suggests that these coiled coil proteinsplay a fundamental role in the organisation and/or function of this highly dynamic Golgicompartment. Furthermore, both the yeast and protozoan GRIP domains function inmammalian cells (Kjer-Nielsen et al., 1999a; McConville et al., 2002), indicating that thetargeting mechanism for the GRIP domain has been highly conserved throughout eukaryoticevolution.

5.3. Targeting to apical and basolateral membranes in polarised cells

Polarity in epithelial cells is determined by the specific localisation of apical and basolateralmembrane proteins to distinct membrane domains. A number of sorting signals have beenidentified that direct proteins to either the apical or basolateral membrane (Nelson and Yeaman,2001). Transport to apical and basolateral membranes appears to be hierarchical, with basolateralsignals having precedence over apical signals and apical transport occurring only in the absence ofa basolateral signal (Matter and Mellman, 1994). In many polarised epithelial cells apical andbasolateral proteins are segregated in the TGN and exit the TGN in distinct transport carriers(Keller et al., 2001). Both sorting at the TGN and recycling from endosomes are important forsteady state localisation at apical and basolateral membranes (Nelson and Yeaman, 2001).

Basolateral sorting signals include the tyrosine containing motif YXXF important in clathrinmediated endocytosis (Bonifacino and Dell’Angelica, 1999). In polarised cells, this motif interactswith an epithelial cell specific subunit of AP1, m1B (Folsch et al., 1999). EM studies have shown


that recycling of the transferrin receptor, a basolateral protein that contains an YXXF signal,occurs in clathrin coated vesicles (Futter et al., 1998). A direct interaction between anotherbasolateral YXXF protein, the low density lipoprotein receptor, and AP1/m1B has also beendemonstrated (Folsch et al., 2001). Other basolateral sorting signals include a di-leucine motif(Hunziker and Fumey, 1994; Miranda et al., 2001) which appears to function via a differentmechanism than the YXXF-based motifs (Miranda et al., 2001).

Membrane microdomains enriched in sphingolipids and cholesterol, known as lipid rafts, arealso thought to play a role in apical sorting, although the mechanisms involved are not clear(Simons and Ikonen, 1997). Many apical proteins contain glycosyl-phosphatidylinositol (GPI)-anchors that are necessary for their localisation to the apical membrane (Brown and Rose, 1992).These GPI-anchors are likely to associate with the lipid rafts during transport to the apical surface(Simons and Ikonen, 1997). N- or O-linked glycans in the lumenal domain of the protein may alsopromote apical sorting, possibly by interacting with lectins in the TGN that sort apical proteins(Fiedler et al., 1994). Signals within membrane anchors of transmembrane proteins may also existfor apical membrane targeting (Keller and Simons, 1997).

While selective delivery and recycling of plasma membrane proteins plays a critical role in themaintenance of cellular polarity, retention of proteins at the target membrane is also important.For example, the cytoplasmic tails of several basolateral membrane proteins contain PDZ-bindingdomains that interact with cytosolic PDZ proteins, retaining them at the plasma membrane(Fanning and Anderson, 1999).

6. Endosome protein sorting

The endosomal/lysosomal system of mammalian cells is a highly dynamic trafficking pathwaythat includes membrane transport from both the late Golgi and the plasma membrane. The basicorganisation of the endosomal/lysosomal system of mammalian cells in now fairly well defined: ithas functionally and physically distinct compartments, which include early endosomes, recyclingendosomes, late endosomes, lysosomes and the TGN (Mellman, 1996). The primary function ofendosomes is the sorting and segregation of receptors and ligands, a process necessary for manycellular operations. For example, many receptors are selectively internalised into the earlyendosome from the cell surface via clathrin-mediated endocytosis (Schmid, 1997). Within thisorganelle some receptors, such as epidermal growth factor (EGF) receptor, are sorted based onthe presence or absence of bound ligand, a critical step in the regulation of the signal transductioncascade induced by ligand binding to this receptor at the cell surface (Ceresa and Schmid, 2000).When ligand is present, both the receptor and ligand are transported to a more acidic lateendosomal compartment for degradation, while in the absence of ligand, the receptor is recycledback to the plasma membrane (Futter et al., 1996).

The endosomal system is also the location for certain specialised cellular functions. Forexample, the efficient loading of antigenic peptides onto MHC Class II molecules occurs withinendosomal compartments (Watts, 1997). Once MHC Class II molecules acquire peptides, they aretransported to the cell surface where they present the antigenic peptide to T lymphocytes. Whilethese and other cellular functions are dependent on the correct sorting of proteins within the


various compartments of the endosomal system, the proteins and mechanisms responsible forthese sorting events remain largely undefined.

Ligands and receptors destined for degradation are delivered from the early endosome to thelate endosome. The late endosome contains many internal vesicles whose membranes differ fromthose of the limiting membrane (Kobayashi et al., 2001). For example, these internal membranescontain high levels of lysobisphosphatidic acid (Gruenberg, 2001). For some time there had beenconsiderable debate regarding the relationship of the early to the late endosome (Gruenberg andMaxfield, 1995; Futter et al., 1996); does transport occur between two static compartments byvesicular transport or do early endosomes mature into late endosomes by removal of componentsof the early endosome? In essence this debate essentially focused on the identity of the transportintermediate. However, this issue has become almost irrelevant as, firstly, it is now realised thatthe endosomal system is more complex than previously thought with the existence of additionalcompartments such as the recycling endosome, secondly, trafficking through the endosomalcompartments is highly dynamic and, thirdly, sorting and transport appears to take place at allstages through the pathway.

Material to be degraded is transported from the late endosome to the lytic environment of thelysosome (Rouille et al., 2000). This probably occurs by fusion of the two compartments to form ahybrid organelle (Luzio et al., 2000). In this model the lysosome acts as a storage granule forlysosomal enzymes that contact endocytosed material upon fusion with the late endosome(Griffiths, 1996). Protein degradation takes place in this late endosome-lysosome hybridorganelle. Lysosomes would then re-form from these hybrid organelles by concentration of thelysosomal enzymes coupled with selective removal of endocytic proteins and subsequent fission ofa new lysosome compartment (Luzio et al., 2000).

It is clear that the endosomes are critical sorting compartments. In particular the earlyendosome needs to distribute cargo destined to the plasma membrane, the recycling endosome,the late endosome and the TGN, highlighting the necessity for defined sorting mechanisms.Therefore, currently there is considerable interest in defining the machinery utilised for sortingand trafficking of cargo from endosomes.

6.1. Dissection of endosomal sorting in yeast

Yeast genetics has provided a powerful approach to dissection of membrane transportpathways. This is particularly true for the complex endosomal system, which has been difficult tounravel at the molecular level in mammalian cells. Yeast genetics has begun to provide anunderstanding of the nature of the protein complexes and lipids responsible for cargo selectionand the biogenesis of transport vesicles from endosomes. Genetic screens in yeast have identifiedmore than 50 vacuolar protein sorting (VPS) genes, involved directly or indirectly in vacuolarhydrolase trafficking (Bankaitis et al., 1986; Rothman and Stevens, 1986; Banta et al., 1988;Robinson et al., 1988; Raymond et al., 1992b). Disruption of the genes implicated in this processcauses disruption and missorting of vacuole hydrolases (Bankaitis et al., 1986; Rothman andStevens, 1986; Banta et al., 1988; Robinson et al., 1988; Raymond et al., 1992b). In yeast, vacuolarhydrolases like carboxypeptidase Y (CPY) bind to their affiliated receptor, Vps10p, in the TGNand are conveyed via vesicles to the prevacuolar compartment (equivalent to the mammalian lateendosome) (Bryant and Stevens, 1997; Horazdovsky et al., 1997; Seaman et al., 1997). The


proteolytically processed hydrolases are subsequently transported to the vacuole,whereas, Vps10p returns from the prevacuolar compartment to the Golgi for furtherrounds of CPY transportation (Cooper and Stevens, 1996). Since these membranereceptors need to cycle between two compartments, a retrograde transport mechanism isemployed to return proteins, including Vps10p, for further rounds of cycling (Cooper andStevens, 1996; Seaman et al., 1998). The transport of Vps10p in yeast is considered analogous tothe recycling of the M6P receptors in mammalian cells (Kornfeld and Mellman, 1989; Traub andKornfeld, 1997).

By characterising the vps mutants, yeast Vps26p, Vps29p, Vps35p, Vps5p along with Vps17pwere identified and deemed important for the sorting and retrograde retrieval process of Vps10pback to the TGN (Horazdovsky et al., 1997; Nothwehr and Hindes, 1997; Seaman et al., 1997;Seaman et al., 1998). Seaman et al. (1998) has proposed a model for a ‘‘retromer complex’’ toconsist of two sub-complexes, the inner shell, Vps35p, Vps29p, Vps26p and outer shell of Vps5pand Vps17p. Vps35p, Vps29p and Vps26p were first identified for their role in Vps10p retrieval(Seaman et al., 1997), although now their function has been refined to selecting cargo to beretrieved from the prevacuolar compartment (PVC) (Seaman et al., 1998). Yeast Vps35p directlyassociates with a sorting signal in the cytosolic domain of several cargo proteins, includingVps10p and A-ALP, a model TGN resident protein (Bryant and Stevens, 1997; Nothwehr andHindes, 1997; Nothwehr et al., 2000). These studies imply that Vps35p is the key retromer subunitfor recognising cytosolic retrieval signals in cargo for retrograde recruitment from the endosomesto the TGN (Nothwehr et al., 2000).

Correct retrograde transport of Vps10p is also dependent on Vps5p (Nothwehr and Hindes,1997). By immunofluorescence, Vps5p localises to an endosomal compartment (Nothwehr andHindes, 1997), and further analysis has revealed that this protein associated with Vps35p onvesicular membranes clustered at the rim of the PVC (Seaman et al., 1998). As Vps5p can self-assemble into large macromolecular complexes both in vivo and in vitro, Vps5p may providesome of the mechanical force to drive vesicle formation (Seaman et al., 1998). Vps5p directlyinteracts with Vps17p (Horazdovsky et al., 1997), and together form the outer coat that providesthe structural component to complete the retromer complex and promote vesicle formation(Seaman et al., 1998).

It would be expected that deletion of each retromer subunit would result inidentical morphologies if they all function selectively in only one specifictransport step. Mutations in either VPS5 or VPS17 belong to class B vps mutants whichdisplay severely fragmented vacuoles (Raymond et al., 1992a). In contrast, however, vps29and vps35 are members of class A vps mutants branded with normal vacuole morphology(Banta et al., 1988; Raymond et al., 1992a), whereas vps26 belongs to class F mutantscharacterised by partially fragmented vacuoles (Raymond et al., 1992a). The observedmorphological differences in retromer subunit deletions may reflect additional roles for each ofthe five retromer subunits.

Overall, the five proteins of the retromer complex have the necessary characteristicsto function as a vesicle coat, for retrograde trafficking from the PVC to the TGN.Vps26p, Vps29p and Vps35p form the inner shell for cargo selection concentration, whileVps5p and Vps17p present the properties for subsequent membrane invagination (Seamanet al., 1998).


6.2. Human retromer complex and sorting nexins

A peripheral membrane protein, sorting nexin 1 (SNX1), has been shown to regulate the cellsurface expression of the human EGF receptor (Kurten et al., 1996). SNX1 was identified, via theyeast two-hybrid system, as a protein that interacts with cytoplasmic sequences of the EGFreceptor, sequences which include the tyrosine kinase domain and the adjacent lysosomaltargeting signal. Over-expression of SNX1 in transfected cells resulted in a specific increase in therate of degradation of the EGF receptor when bound to its ligand (Kurten et al., 1996). Haft andco-workers demonstrated a direct interaction between the EGF receptor and SNX1 using co-immunoprecipitation experiments (Haft et al., 1998). In addition, SNX1 has recently been shownto self-associate in vivo (Kurten et al., 2001) and is proposed to form oligomers with other SNX1related proteins.

A relationship has been demonstrated between sorting nexins (SNXs) and the retromercomplex. Sequence homology identified human genes orthologous to those encoding the yeastretromer complex (Haft et al., 2000). Human VPS26, VPS29 and VPS35 are orthologous to theircounterparts in yeast (Nothwehr and Hindes, 1997; Haft et al., 1998), whereas, the SNX family ofproteins in humans are related to VPS5 and VPS17 (Haft et al., 1998; Teasdale et al., 2001).Conservation of the retromer components suggests it is an important constituent in intracellularretrieval from the endosome and may confer an analogous sorting mechanism (Haft et al., 2000).Co-immunoprecipitation experiments have demonstrated antibodies to human Vps26p couldimmunoabsorb human Vps26p, Vps29p, Vps35p and SNX1 (Haft et al., 2000). A number of theSNXs display an early endosomal localisation (Parks et al., 2001; Teasdale et al., 2001; Zhonget al., 2002) and moreover, the localisation of SNX1 has been further refined to sorting endosomes(Kurten et al., 2001). Endogenous SNX1 and SNX2 show strong co-localisation with each other(Zhong et al., 2002) and co-localise with GFP-tagged-human Vps35p in vivo (E. Thomas, M. Kerrand R. D. Teasdale, unpublished observations). This suggests the formation of a multimeric‘‘human retromer complex’’ in mammalian cells, consisting of yeast retromer subunit orthologsand sorting nexins. As SNX1 interacts with EGF receptor, it represents a good candidate forsorting cargo proteins within the early endosome for the subsequent membrane transport stepdestined to the late endosome (Haft et al., 1998).

SNX1 is one of 22 identified members of the SNX family, characterised by the presence of a70–110 amino acid PX domain (Teasdale et al., 2001), a domain which allows SNXs to bind thelipids phosphatidylinositol-3-phosphate (PI(3)P) (Xu et al., 2001; Cozier et al., 2002) andphosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) (Cozier et al., 2002). PX domains (Ponting,1996) are present in many yeast/human membrane and vesicle-associated proteins involved in avariety of cell signalling pathways (Schultz et al., 2000). Proteins can associate intimately andspecifically to membrane compartments through their PX domain (Schultz et al., 2000), bybinding phosphoinositol lipids present in the membrane (Wishart et al., 2001). As the earlyendosome is enriched in PI(3)P, an interaction with this membrane lipid enables specificlocalisation to this compartment (Gillooly et al., 2000). Nonetheless, considerably more work isrequired to ascertain the precise endosomal localisation of the different SNX family members. Fora recent detailed review on SNXs see Worby and Dixon (2002).

The selectivity for particular cargo by the retromer complex may arise from the properties ofSNX molecules (Haft et al., 1998; Kurten et al., 2001). Homo and hetero-oligomers of SNX1,


SNX2 and SNX4, and heteromeric interactions of these SNXs with SNX6, have been observed. Itis interesting to speculate that the formation of a heterotetramer of different SNXs could functionto recruit a range of cargo proteins to a single retromer complex. Immunoprecipitationexperiments have demonstrated that SNXs can interact/associate with a variety of plasmamembrane receptors, including several tyrosine kinase receptors, platelet derived growth factorreceptor, insulin receptor and transferrin receptor (Kurten et al., 1996; Haft et al., 1998). Thus,each SNX may bind distinct subsets of cargo proteins within the endosomal compartments andrecruit them into defined transport carriers for a specific destination. The considerable currentinterest in the SNX molecules should ensure that their precise role(s) in the sorting events of theendosomal pathway are clarified in the near future.

7. Conclusion

The secretory and endocytic pathways of eukaryotic cells consist of multiple organelles withspecialised functions mediated by their resident proteins. Localisation of proteins to specificmembranes is complex and often involves multiple interactions. Specific targeting signals withinresident proteins interact with proteins and/or lipids to enable them to localise to unique sites inthe cell. In addition, many proteins involved in membrane transport cycle between compartmentsin transport carriers. These proteins also require signals to ensure loading onto the correcttransport carriers and recycling back to the original compartment.

As biochemical assays become more sensitive and genetic screens more sophisticated, additionalproteins involved in vesicle transport are rapidly being identified. The application ofbioinformatics has led to the discovery of many novel proteins and protein families involved inprotein sorting and membrane transport. Our understanding of the processes involved inmembrane trafficking is expanding rapidly as divergent pieces merge to form a morecomprehensive picture. The ultimate challenge is to fully define the molecular steps that regulatethe selective movement of proteins and lipids throughout the cell.

Acknowledgements

Funding from the Australian National Health and Medical Research Council, and theAustralian Research Council is gratefully acknowledged. Dr. Ana Merino-Trigo was supportedby a post-doctoral Fellowship from the Ministerio de Cultura, Educaci !on y Ciencia.

References

Aalto, M.K., Keranen, S., Ronne, H., 1992. A family of proteins involved in intracellular transport. Cell 68, 181–182.

Allan, B.B., Moyer, B.D., Balch, W.E., 2000. Rab1 recruitment of p115 into a cis-SNARE complex: programming

budding COPII vesicles for fusion. Science 289, 444–448.

Alvarez, C., Fujita, H., Hubbard, A., Sztul, E., 1999. ER to Golgi transport: requirement for p115 at a pre-Golgi VTC

stage. J. Cell Biol. 147, 1205–1221.


Andersson, H., Kappeler, F., Hauri, H.P., 1999. Protein targeting to endoplasmic reticulum by dilysine signals involves

direct retention in addition to retrieval. J. Biol. Chem. 274, 15080–15084.

Antonny, B., Madden, D., Hamamoto, S., Orci, L., Schekman, R., 2001. Dynamics of the COPII coat with GTP and

stable analogues. Nat. Cell Biol. 3, 531–537.

Aridor, M., Hannan, L.A., 2000. Traffic jam: a compendium of human diseases that affect intracellular transport

processes. Traffic 1, 836–851.

Aridor, M., Bannykh, S.I., Rowe, T., Balch, W.E., 1995. Sequential coupling between COPII and COPI vesicle coats in

endoplasmic reticulum to Golgi transport. J. Cell Biol. 131, 875–893.

Baeuerle, P.A., Huttner, W.B., 1987. Tyrosine sulfation is a trans-Golgi-specific protein modification. J. Cell Biol. 105,

2655–2664.

Bankaitis, V.A., 2002. Cell biology. Slick recruitment to the Golgi. Science 295, 290–291.

Bankaitis, V.A., Johnson, L.M., Emr, S.D., 1986. Isolation of yeast mutants defective in protein targeting to the

vacuole. Proc. Natl. Acad. Sci. USA 83, 9075–9079.

Bannykh, S.I., Rowe, T., Balch, W.E., 1996. The organization of endoplasmic reticulum export complexes. J. Cell Biol.

135, 19–35.

Banta, L.M., Robinson, J.S., Klionsky, D.J., Emr, S.D., 1988. Organelle assembly in yeast: characterization of yeast

mutants defective in vacuolar biogenesis and protein sorting. J. Cell Biol. 107, 1369–1383.

Barlowe, C., 1998. COPII and selective export from the endoplasmic reticulum. Biochim. Biophys. Acta 1404,

67–76.

Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazzola, M., Amherdt,

M., Schekman, R., 1994. COPII: a membrane coat formed by sec proteins that drive vesicle budding from the

endoplasmic reticulum. Cell 77, 895–907.

Barlowe, C., Schekman, R., 1993. SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle

budding from the ER. Nature 365, 347–349.

Baron, C.L., Malhotra, V., 2002. Role of diacylglycerol in PKD recruitment to the TGN and protein transport to the

plasma membrane. Science 295, 325–328.

Barr, F.A., 1999. A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins. Curr. Biol. 9,

381–384.

Barr, F.A., Nakamura, N., Warren, G., 1998. Mapping the interaction between GRASP65 and GM130, components of

a protein complex involved in the stacking of Golgi cisternae. EMBO J. 17, 3258–3268.

Barr, F.A., Puype, M., Vandekerckhove, J., Warren, G., 1997. Grasp65, a protein involved in the stacking of Golgi

cisternae. Cell 91, 253–262.

Belden, W.J., Barlowe, C., 2001. Role of Erv29P in collecting soluble secretory proteins into er-derived transport

vesicles. Science 294, 1528–1531.

Berger, E.G., 1998. Camillo Golgi. Trends Cell Biol. 8, 1.

Block, M.R., Glick, B.S., Wilcox, C.A., Wieland, F.T., Rothman, J.E., 1988. Purification of an N-ethylmaleimide-

sensitive protein catalyzing vesicular transport. Proc. Natl. Acad. Sci. USA 85, 7852–7856.

Boman, A.L., 2001. GGA proteins: New players in the sorting game. J. Cell Sci. 114, 3413–3418.

Boman, A.L., Zhang, C.J., Zhu, X.J., Kahn, R.A., 2000. A family of ADP-ribosylation factor effectors that can alter

membrane transport through the trans-Golgi. Mol. Biol. Cell 11, 1241–1255.

Bonfanti, L., Mironov Jr., A.A., Martinez-Menarguez, J.A., Martella, O., Fusella, A., Baldassarre, M., Buccione, R.,

Geuze, H.J., Mironov, A.A., Luini, A., 1998. Procollagen traverses the Golgi stack without leaving the lumen of

cisternae: evidence for cisternal maturation. Cell 95, 993–1003.

Bonifacino, J.S., Dell’Angelica, E.C., 1999. Molecular bases for the recognition of tyrosine-based sorting signals. J. Cell

Biol. 145, 923–926.

Bos, K., Wraight, C., Stanley, K.K., 1993. TGN38 is maintained in the trans-Golgi network by a tyrosine-containing

motif in the cytoplasmic domain. EMBO J. 12, 2219–2228.

Bremser, M., Nickel, W., Schweikert, M., Ravazzola, M., Amherdt, M., Hughes, C.A., Sollner, T.H., Rothman, J.E.,

Wieland, F.T., 1999. Coupling of coat assembly and vesicle budding to packaging of putative cargo receptors. Cell

96, 495–506.

Bretscher, M.S., Munro, S., 1993. Cholestrol and Golgi apparatus. Science 261, 1280–1281.


Broadie, K., Prokop, A., Bellen, H.J., O’Kane, C.J., Schulze, K.L., Sweeney, S.T., 1995. Syntaxin and synaptobrevin

function downstream of vesicle docking in Drosophila. Neuron 15, 663–673.

Brockhausen, I., 1995. Biosynthesis of O-glycans of the N-acetylgalactosamine-a-Ser/Thr linkage type. In:

Montreuil, J., Vliegenthart, J., Schachter, H. (Eds.), New Comprehensive Biochemistry, Vol. 29a. Elsevier,

Amsterdam.

Brown, D.L., Heimann, K., Lock, J., Kjer-Nielsen, L., van Vliet, C., Stow, J.L., Gleeson, P.A., 2001. The GRIP

domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers.

Traffic 2, 336–344.

Brown, D.A., Rose, J.K., 1992. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during

transport to the apical cell surface. Cell 68, 533–544.

Brown, P.S., Wang, E., Aroeti, B., Chapin, S.J., Mostov, K.E., Dunn, K.W., 2000. Definition of distinct compartments

in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical

recycling. Traffic 1, 124–140.

Bryant, N.J., James, D.E., 2001. Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE

complex formation. EMBO J. 20, 3380–3388.

Bryant, N.J., Stevens, T.H., 1997. Two separate signals act independently to localize a yeast late Golgi membrane

protein through a combination of retrieval and retention. J. Cell Biol. 136, 287–297.

Burd, C.G., Emr, S.D., 1998. Phosphatidylinositol(3)-phosphate signaling mediated by specific binding to RING

FYVE domains. Mol. Cell 2, 157–162.

Burgess, T.L., Kelly, R.B., 1987. Constitutive and regulated secretion of proteins. Annu. Rev. Cell Biol. 3, 243–293.

Burke, J., Pettitt, J.M., Humphris, D., Gleeson, P.A., 1994. Medial-Golgi retention of N-acetylglucosaminyltransferase

I—contribution from all domains of the enzyme. J. Biol. Chem. 269, 12049–12059.

Burke, J., Pettitt, J.M., Schachter, H., Sarkar, M., Gleeson, P.A., 1992. The transmembrane and flanking sequences of

b1,2-N-acetylglucosaminyltransferase I specify medial-Golgi localization. J. Biol. Chem. 267, 24433–24440.

Callaghan, J., Simonsen, A., Gaullier, J.M., Toh, B.H., Stenmark, H., 1999. The endosome fusion regulator early-

endosomal autoantigen 1 (EEA1) is a dimer. Biochem. J. 338, 539–543.

Cao, X., Ballew, N., Barlowe, C., 1998. Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is

independent of SNARE proteins. EMBO J. 17, 2156–2165.

Ceresa, B.P., Schmid, S.L., 2000. Regulation of signal transduction by endocytosis. Curr. Opin. Cell Biol. 12, 204–210.

Chardin, P., McCormick, F., 1999. Brefeldin A: The advantage of being uncompetitive. Cell 97, 153–155.

Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C.L., Chabre, M., 1996. A human

exchange factor for ARF contains Sec7- and pleckstrin-homology domains. Nature 384, 481–484.

Chavrier, P., Goud, B., 1999. The role of ARF and Rab GTPases in membrane transport. Curr. Opin. Cell Biol. 11,

466–475.

Chege, N.W., Pfeffer, S.R., 1990. Compartmentation of the Golgi complex: brefeldin-A distinguishes trans-Golgi

cisternae from the trans-Golgi network. J. Cell Biol. 111, 893–899.

Chen, C., Ma, J.Y., Lazic, A., Backovic, M., Colley, K.J., 2000. Formation of insoluble oligomers correlates with

ST6Gal I stable localization in the Golgi. J. Biol. Chem. 275, 13819–13826.

Christoforidis, S., McBride, H.M., Burgoyne, R.D., Zerial, M., 1999a. The rab5 effector EEA1 is a core component of

endosome docking. Nature 397, 621–625.

Christoforidis, S., Miaczynska, M., Ashman, K., Wilm, M., Zhao, L., Yip, S.C., Waterfield, M.D., Backer, J.M.,

Zerial, M., 1999b. Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nat. Cell Biol. 1, 249–252.

Clary, D.O., Griff, I.C., Rothman, J.E., 1990. SNAPs, a family of NSF attachment proteins involved in intracellular

membrane fusion in animals and yeast. Cell 61, 709–721.

Cole, N.B., Ellenberg, J., Song, J., DiEuliis, D., Lippincott-Schwartz, J., 1998. Retrograde transport of Golgi-localized

proteins to the ER. J. Cell Biol. 140, 1–15.

Cole, N.B., Lippincott-Schwartz, J., 1995. Organization of organelles and membrane traffic by microtubules. Curr.

Opin. Cell Biol. 7, 55–64.

Colley, K.J., 1997. Golgi localization of glycosyltransferases: more questions than answers. Glycobiology 7, 1–13.

Cooper, A.A., Stevens, T.H., 1996. Vps10p cycles between the late-Golgi and prevacuolar compartments in its function

as the sorting receptor for multiple yeast vacuolar hydrolases. J. Cell Biol. 133, 529–541.


Cosson, P., Letourneur, F., 1994. Coatomer interaction with di-lysine endoplasmic reticulum retention motifs. Science

263, 1629–1631.

Cozier, G.E., Carlton, J., McGregor, A.H., Gleeson, P.A., Teasdale, R.D., Mellor, H., Cullen, P.J., 2002. The PX

domain-dependent, 3-phosphoinositide-mediated association of sorting nexin-1 with an early sorting endosomal

compartment is required for its ability to regulate epidermal growth factor receptor degradation. J. Biol. Chem. 277,

48730–48736.

Cukierman, E., Huber, I., Rotman, M., Cassel, D., 1995. The ARF1 GTPase-activating protein: zinc finger motif and

Golgi complex localization. Science 270, 1999–2002.

Dalton, A., Felix, M., 1954. Cytologic and cytochemical characteristics of the Golgi substance of epithelial cells of the

epididymis-in situ, in homogenates and after isolation. Am. J. Anat. 94, 171–187.

Dell’Angelica, E.C., Puertollano, R., Mullins, C., Aguilar, R.C., Vargas, J.D., Hartnell, L.M., Bonifacino, J.S., 2000.

GGAs: a family of ADP ribosylation factor-binding proteins related to adaptors and associated with the Golgi

complex. J. Cell Biol. 149, 81–93.

Dirac-Svejstrup, A.B., Shorter, J., Waters, M.G., Warren, G., 2000. Phosphorylation of the vesicle-tethering protein

p115 by a casein kinase II-like enzyme is required for Golgi reassembly from isolated mitotic fragments. J. Cell Biol.

150, 475–487.

Dittie, A.S., Thomas, L., Thomas, G., Tooze, S.A., 1997. Interaction of furin in immature secretory granules from

neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation. EMBO J. 16,

4859–4870.

Do, H., Falcone, D., Lin, J., Andrews, D.W., Johnson, A.E., 1996. The cotranslational integration of membrane

proteins into the phospholipid bilayer is a multistep process. Cell 85, 369–378.

Donaldson, J.G., Lippincott-Schwartz, J., Bloom, G.S., Kreis, T.E., Klausner, R.D., 1990. Dissociation of a 110-kD

peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J. Cell Biol. 111, 2295–2306.

Fanning, A.S., Anderson, J.M., 1999. Protein modules as organizers of membrane structure. Curr. Opin. Cell Biol. 11,

432–439.

Farquhar, M.G., 1985. Progress in unraveling pathways of Golgi traffic. Annu. Rev. Cell Biol. 1, 447–488.

Farquhar, M.G., Palade, G.E., 1981. The Golgi apparatus (complex)—(1954–1981)—from artifact to centre stage.

J. Cell Biol. 91, 77s–103s.

Fiedler, K., Parton, R.G., Kellner, R., Etzold, T., Simons, K., 1994. Vip36, a novel component of glycolipid rafts and

exocytic carrier vesicles in epithelial cells. EMBO J. 13, 1729–1740.

Fiedler, K., Veit, M., Stamnes, M.A., Rothman, J.E., 1996. Bimodal interaction of coatomer with the p24 family of

putative cargo receptors. Science 273, 1396–1399.

Folsch, H., Ohno, H., Bonifacino, J.S., Mellman, I., 1999. A novel clathrin adaptor complex mediates basolateral

targeting in polarized epithelial cells. Cell 99, 189–198.

Folsch, H., Pypaert, M., Schu, P., Mellman, I., 2001. Distribution and function of AP-1 clathrin adaptor complexes in

polarized epithelial cells. J. Cell Biol. 152, 595–606.

Ford, M.G., Pearse, B.M., Higgins, M.K., Vallis, Y., Owen, D.J., Gibson, A., Hopkins, C.R., Evans, P.R., McMahon,

H.T., 2001. Simultaneous binding of PtdIns(4,5)P2 and clathrin by AP180 in the nucleation of clathrin lattices on

membranes. Science 291, 1051–1055.

Francis, M.J., Jones, E.E., Levy, E.R., Ponnambalam, S., Chelly, J., Monaco, A.P., 1998. A Golgi localization signal

identified in the Menkes recombinant protein. Hum. Mol. Genet. 7, 1245–1252.

Francis, M.J., Jones, E.E., Levy, E.R., Martin, R.L., Ponnambalam, S., Monaco, A.P., 1999. Identification of a di-

leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the

plasma membrane. J Cell Sci. 112, 1721–1732.

Freedman, R.B., 1994. Protein folding. Folding helpers and unhelpful folders. Curr. Biol. 4, 933–935.

Friend, D.S., Murray, M.J., 1965. Am. J. Anat. 117, 135–149.

Futerman, A.H., Stieger, B., Hubbard, A.L., Pagano, R.E., 1990. Sphingomyelin synthesis in rat liver occurs

predominantly at the cis and medial cisternae of the Golgi apparatus. J. Biol. Chem. 265, 8650–8657.

Futter, C.E., Gibson, A., Allchin, E.H., Maxwell, S., Ruddock, L.J., Odorizzi, G., Domingo, D., Trowbridge, I.S.,

Hopkins, C.R., 1998. In polarized MDCK cells basolateral vesicles arise from clathrin-gamma-adaptin-coated

domains on endosomal tubules. J. Cell Biol. 141, 611–623.


Futter, C.E., Pearse, A., Hewlett, L.J., Hopkins, C.R., 1996. Multivesicular endosomes containing internalized EGF–

EGF receptor complexes mature and then fuse directly with lysosomes. J. Cell Biol. 132, 1011–1023.

Garcia, E.P., McPherson, P.S., Chilcote, T.J., Takei, K., De Camilli, P., 1995. RbSec1A and B colocalize with syntaxin

1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin. J. Cell Biol. 129, 105–120.

Gething, M.J., 1999. Role and regulation of the ER chaperone BiP. Semin. Cell Dev. Biol. 10, 465–472.

Gillooly, D.J., Morrow, I.C., Lindsay, M., Gould, R., Bryant, N.J., Gaullier, J.M., Parton, R.G., Stenmark, H., 2000.

Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19, 4577–4588.

Giraudo, C.G., Daniotti, J.L., Maccioni, H.J., 2001. Physical and functional association of glycolipid N-acetyl-

galactosaminyl and galactosyl transferases in the Golgi apparatus. Proc. Natl. Acad. Sci. USA 98, 1625–1630.

Girotti, M., Banting, G., 1996. TGN38-green fluorescent protein hybrid proteins expressed in stably transfected

eukaryotic cells provide a tool for the real-time, in vivo study of membrane traffic pathways and suggest a possible

role for ratTGN38. J. Cell Sci. 109, 2915–2926.

Gleeson, P.A., 1998. Targeting of proteins to the Golgi apparatus. Histochem. Cell Biol. 109, 517–532.

Gleeson, P.A., Anderson, T.J., Stow, J.L., Griffiths, G., Toh, B.H., Matheson, F., 1996. P230 is associated with vesicles

budding from the trans-Golgi network. J. Cell Sci. 109, 2811–2821.

Glick, B.S., Elston, T., Oster, G., 1997. A cisternal maturation mechanism can explain the asymmetry of the Golgi

stack. FEBS Lett. 414, 177–181.

Goldberg, J., 1999. Structural and functional analysis of the ARF1-ARFGAP complex reveals a role for coatomer in

GTP hydrolysis. Cell 96, 893–902.

Golgi, C., 1898. Sur la structure des cellules nerveuses. Arch. Ital. Biol. 30, 60–71.

Gonzalez Jr., L., Scheller, R.H., 1999. Regulation of membrane trafficking: structural insights from a Rab/effector

complex. Cell 96, 755–758.

Goode, B.L., Drubin, D.G., Barnes, G., 2000. Functional cooperation between the microtubule and actin

cytoskeletons. Curr. Opin. Cell Biol. 12, 63–71.

Gorlich, D., Rapoport, T.A., 1993. Protein translocation into proteoliposomes reconstituted from purified components

of the endoplasmic reticulum membrane. Cell 75, 615–630.

Graham, T.R., Krasnov, V.A., 1995. Sorting of yeast alpha 1,3 mannosyltransferase is mediated by a lumenal domain

interaction, and a transmembrane domain signal that can confer clathrin-dependent Golgi localization to a secreted

protein. Mol. Biol. Cell 6, 809–824.

Griffiths, G., 1996. On vesicles and membrane compartments. Protoplasma 195, 37–58.

Griffiths, G., 2000. Gut thoughts on the Golgi complex. Traffic 1, 738–745.

Griffiths, G., Simons, K., 1986. The trans Golgi network: sorting at the exit site of the golgi complex. Science 234,

438–443.

Gruenberg, J., 2001. The endocytic pathway: A mosaic of domains. Nat. Rev. Mol. Cell Biol. 2, 721–730.

Gruenberg, J., Maxfield, F.R., 1995. Membrane transport in the endocytic pathway. Curr. Opin. Cell Biol. 7, 552–563.

Haft, C.R., de la Luz Sierra, M., Bafford, R., Lesniak, M.A., Barr, V.A., Taylor, S.I., 2000. Human orthologs of yeast

vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes. Mol. Biol. Cell 11,

4105–4116.

Haft, C.R., de la Luz Sierra, M., Barr, V.A., Haft, D.H., Taylor, S.I., 1998. Identification of a family of sorting nexin

molecules and characterization of their association with receptors. Mol. Cell Biol. 18, 7278–7287.

Hammond, C., Helenius, A., 1995. Quality control in the secretory pathway. Curr. Opin. Cell Biol. 7, 523–529.

Hauri, H.P., Schweizer, A., 1992. The endoplasmic reticulum-Golgi intermediate compartment. Curr. Opin. Cell Biol.

4, 600–608.

Helenius, A., 1994. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol. Biol.

Cell 5, 253–265.

Helenius, A., Trombetta, E., Herbert, D., Simons, J., 1997. Calnexin, calreticulin and the folding of glycoproteins.

Trends Cell Biol. 7, 193–200.

Herrmann, J.M., Malkus, P., Schekman, R., 1999. Out of the ER—outfitters, escorts and guides. Trends Cell Biol. 9,

5–7.

Hinshaw, J.E., Schmid, S.L., 1995. Dynamin self-assembles into rings suggesting a mechanism for coated vesicle

budding. Nature 374, 190–192.


Hirschberg, K., Miller, C.M., Ellenberg, J., Presley, J.F., Siggia, E.D., Phair, R.D., Lippincott-Schwartz, J., 1998.

Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport

intermediates in living cells. J. Cell Biol. 143, 1485–1503.

Hirst, J., Lui, W.W.Y., Bright, N.A., Totty, N., Seaman, M.N.J., Robinson, M.S., 2000. A family of proteins with

gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/

lysosome. J. Cell Biol. 149, 67–79.

Holthuis, J.C., Pomorski, T., Raggers, R.J., Sprong, H., Van Meer, G., 2001. The organizing potential of sphingolipids

in intracellular membrane transport. Physiol. Rev. 81, 1689–1723.

Horazdovsky, B.F., Davies, B.A., Seaman, M.N., McLaughlin, S.A., Yoon, S., Emr, S.D., 1997. A sorting nexin-1

homologue, Vps5p, forms a complex with Vps17p and is required for recycling the vacuolar protein-sorting receptor.

Mol. Biol. Cell 8, 1529–1541.

Hsu, S.C., Ting, A.E., Hazuka, C.D., Davanger, S., Kenny, J.W., Kee, Y., Scheller, R.H., 1996. The mammalian brain

rsec6/8 complex. Neuron 17, 1209–1219.

Humphrey, J.S., Peters, P.J., Yuan, L.C., Bonifacino, J.S., 1993. Localization of TGN38 to the trans-Golgi network:

involvement of a cytoplasmic tyrosine-containing sequence. J. Cell Biol. 120, 1123–1135.

Hunziker, W., Fumey, C., 1994. A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc

receptors in MDCK cells. EMBO J. 13, 2963–2967.

Hunziker, W., Geuze, H.J., 1996. Intracellular trafficking of lysosomal membrane proteins. Bioessays 18, 379–389.

Infante, C., Ramos-Morales, F., Fedriani, C., Bornens, M., Rios, R.M., 1999. GMAP-210, A cis-Golgi network-

associated protein, is a minus end microtubule-binding protein. J. Cell Biol. 145, 83–98.

Jackson, T., 1998. Transport vesicles: Coats of many colours. Curr. Biol. 8, R609–R612.

Jackson, M.R., Nilsson, T., Peterson, P.A., 1993. Retrieval of transmembrane proteins to the endoplasmic reticulum.

J. Cell Biol. 121, 317–333.

Jeckel, D., Karrenbauer, A., Birk, R., Schmidt, R.R., Wieland, F., 1990. Sphingomyelin is synthesized in the cis Golgi.

FEBS Lett. 261, 155–157.

Jones, B.G., Thomas, L., Molloy, S.S., Thulin, C.D., Fry, M.D., Walsh, K.A., Thomas, G., 1995. Intracellular

trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail. EMBO

J. 14, 5869–5883.

Jungmann, J., Munro, S., 1998. Multi-protein complexes in the cis Golgi of Saccharomyces cerevisiae with alpha-1,6-

mannosyltransferase activity. EMBO J. 17, 423–434.

Jungmann, J., Rayner, J.C., Munro, S., 1999. The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a

Golgi mannosyltransferase complex. J. Biol. Chem. 274, 6579–6585.

Kaiser, C., 2000. Thinking about p24 proteins and how transport vesicles select their cargo. Proc. Natl. Acad. Sci. USA

97, 3783–3785.

Kaiser, C.A., Schekman, R., 1990. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the

secretory pathway. Cell 61, 723–733.

Kamal, A., Goldstein, L.S., 2000. Connecting vesicle transport to the cytoskeleton. Curr. Opin. Cell Biol. 12, 503–508.

Kanaseki, T., Kadota, K., 1969. The ‘‘vesicle in a basket’’. A morphological study of the coated vesicle isolated from

the nerve endings of the Guinea pig brain, with special reference to the mechanism of membrane movements. J. Cell

Biol. 42, 202–220.

Kelleher, J.F., Titus, M.A., 1998. Intracellular motility: how can we all work together? Curr. Biol. 8, R394–R397.

Keller, P., Simons, K., 1997. Post-Golgi biosynthetic trafficking. J. Cell Sci. 110, 3001–3009.

Keller, P., Toomre, D., Diaz, E., White, J., Simons, K., 2001. Multicolour imaging of post-Golgi sorting and trafficking

in live cells. Nat. Cell Biol. 3, 140–149.

Kirchhausen, T., 1999. Adaptors for clathrin-mediated traffic. Ann. Rev. Cell Dev. Biol. 15, 705–732.

Kjer-Nielsen, L., Teasdale, R.D., van Vliet, C., Gleeson, P.A., 1999a. A novel Golgi-localisation domain shared by a

class of coiled-coil peripheral membrane proteins. Curr. Biol. 9, 385–388.

Kjer-Nielsen, L., van Vliet, C., Erlich, R., Toh, B.H., Gleeson, P.A., 1999b. The Golgi-targeting sequence of the

peripheral membrane protein p230. J. Cell Sci. 112, 1645–1654.

Klumperman, J., 2000. Transport between ER and Golgi. Curr. Opin. Cell Biol. 12, 445–449.


Kobayashi, T., Yamaji-Hasegawa, A., Kiyokawa, E., 2001. Lipid domains in the endocytic pathway. Semin. Cell Dev.

Biol. 12, 173–182.

Kornfeld, R., Kornfeld, S., 1985. Assembly of asparagine-linked oligosaccharides. Ann. Rev. Biochem. 54, 631–664.

Kornfeld, S., Mellman, I., 1989. The biogenesis of lysosomes. Annu. Rev. Cell Biol. 5, 483–525.

Kuehn, M.J., Schekman, R., 1997. CopII and secretory cargo capture into transport vesicles. Curr. Opin. Cell Biol. 9,

477–483.

Kurten, R.C., Cadena, D.L., Gill, G.N., 1996. Enhanced degradation of EGF receptors by a sorting nexin, SNX1.

Science 272, 1008–1010.

Kurten, R.C., Eddington, A.D., Chowdhury, P., Smith, R.D., Davidson, A.D., Shank, B.B., 2001. Self-assembly and

binding of a sorting nexin to sorting endosomes. J. Cell Sci. 114, 1743–1756.

Kutateladze, T.G., Ogburn, K.D., Watson, W.T., de Beer, T., Emr, S.D., Burd, C.G., Overduin, M., 1999.

Phosphatidylinositol 3-phosphate recognition by the FYVE domain. Mol. Cell 3, 805–811.

Ladinsky, M.S., Mastronarde, D.N., McIntosh, J.R., Howell, K.E., Staehelin, L.A., 1999. Golgi structure in three

dimensions: Functional insights from the normal rat kidney cell. J. Cell Biol. 144, 1135–1149.

Lannert, H., Gorgas, K., Meissner, I., Wieland, F.T., Jeckel, D., 1998. Functional organization of the Golgi apparatus

in glycosphingolipid biosynthesis. Lactosylceramide and subsequent glycosphingolipids are formed in the lumen of

the late Golgi. J. Biol. Chem. 273, 2939–2946.

Lanoix, J., Ouwendijk, J., Lin, C.-C., Stark, A., Love, H.D., Ostermann, J., Nilsson, T., 1999. GTP hydrolysis by arf-1

mediates sorting and concentration of Golgi resident enzymes into functional COP I vesicles. EMBO J. 18, 4935–

4948.

Lanoix, J., Ouwendijk, J., Stark, A., Szafer, E., Cassel, D., Dejgaard, K., Weiss, M., Nilsson, T., 2001. Sorting of

Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1. J. Cell Biol. 155,

1199–1212.

Le Borgne, R., Griffiths, G., Hoflack, B., 1996. Mannose 6-phosphate receptors and ADP-ribosylation factors

cooperate for high affinity interaction of the AP-1 Golgi assembly proteins with membranes. J. Biol. Chem. 271,

2162–2170.

Le Borgne, R., Hoflack, B., 1998. Protein transport from the secretory to the endocytic pathway in mammalian cells.

Biochim. Biophys. Acta 1404, 195–209.

Letourneur, F., Gaynor, E.C., Hennecke, S., Demolliere, C., Duden, R., Emr, S.D., Riezman, H., Cosson, P., 1994.

Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum. Cell 79, 1199–1207.

Levine, T.P., Munro, S., 1998. The pleckstrin homology domain of oxysterol-binding protein recognises a determinant

specific to Golgi membranes. Curr. Biol. 8, 729–739.

Levine, T.P., Munro, S., 2002. Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-Kinase-

dependent and -independent components. Curr. Biol. 12, 695–704.

Lewis, M.J., Pelham, H.R., 1990. A human homologue of the yeast HDEL receptor. Nature 348, 162–163.

Lewis, M.J., Pelham, H.R., 1992. Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to

the endoplasmic reticulum. Cell 68, 353–364.

Linstedt, A.D., Hauri, H.P., 1993. Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic

domain of at least 350 kDa. Mol. Biol. Cell 4, 679–693.

Lippincott-Schwartz, J., 1998. Cytoskeletal proteins and Golgi dynamics. Curr. Biol. 10, 52–59.

Lippincott-Schwartz, J., Roberts, T.H., Hirschberg, K., 2000. Secretory protein trafficking and organelle dynamics in

living cells. Annu. Rev. Cell Dev. Biol. 16, 557–589.

Lord, J.M., Davey, J., Frigerio, L., Roberts, L.M., 2000. Endoplasmic reticulum-associated protein degradation.

Semin. Cell Dev. Biol. 11, 159–164.

Love, H.D., Lin, C.-C., Short, C.S., Osterman, J., 1998. Isolation of functional Golgi-derived vesicles with a possible

role in retrograde transport. J. Cell Biol. 140, 541–551.

Lowe, M., 2000. Membrane transport: tethers and TRAPPS. Curr. Biol. 10, R407–R409.

Lupashin, V.V., Pokrovskaya, I.D., McNew, J.A., Waters, M.G., 1997. Characterization of a novel yeast SNARE

protein implicated in Golgi retrograde traffic. Mol. Biol. Cell 8, 2659–2676.

Luzio, J.P., Brake, B., Banting, G., Howell, K.E., Braghetta, P., Stanley, K.K., 1990. Identification, sequencing and

expression of an integral membrane protein of the trans-Golgi network (TGN38). Biochem. J. 270, 97–102.


Luzio, J.P., Rous, B.A., Bright, N.A., Pryor, P.R., Mullock, B.M., Piper, R.C., 2000. Lysosome-endosome fusion and

lysosome biogenesis. J. Cell Sci. 113, 1515–1524.

Ma, J.Y., Qian, R., Rausa, F.M., Colley, K.J., 1997. Two naturally occurring alpha 2,6-sialyltransferase forms with a

single amino acid change in the catalytic domain differ in their catalytic activity and proteolytic processing. J. Biol.

Chem. 272, 672–679.

Machamer, C.E., 1991. Golgi retention signals: Do membranes hold the key? Trends Cell Biol. 1, 141–144.

Maeda, Y., Beznoussenko, G.V., Van Lint, J., Mironov, A.A., Malhotra, V., 2001. Recruitment of protein kinase D to

the trans-Golgi network via the first cysteine-rich domain. EMBO J. 20, 5982–5990.

Malhotra, V., Serafini, T., Orci, L., Shepherd, J.C., Rothman, J.E., 1989. Purification of a novel class of coated vesicles

mediating biosynthetic protein transport through the Golgi stack. Cell 58, 329–336.

Marsh, M., McMahon, H.T., 1999. The structural era of endocytosis. Science 285, 215–220.

Marth, J., 1996. Complexity in O-linked oligosacharide biosynthesis engendered by multiple polypeptide N-

acetylgalactosaminyltransferases. Glycobiology 6, 701–705.

Martinez-Menarguez, J.A., Geuze, H.J., Slot, J.W., Klumperman, J., 1999. Vesicular tubular clusters between the ER

and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles. Cell 98,

81–90.

Martinez-Menarguez, J.A., Prekeris, R., Oorschot, V.M., Scheller, R., Slot, J.W., Geuze, H.J., Klumperman, J., 2001.

Peri-Golgi vesicles contain retrograde but not anterograde proteins consistent with the cisternal progression model

of intra-Golgi transport. J. Cell Biol. 155, 1213–1224.

Martoglio, B., Dobberstein, B., 1998. Signal sequences: More than just greasy peptides. Trends Cell Biol. 8, 410–415.

Masibay, A.S., Balaji, P.V., Boeggeman, E.E., Qasba, P.K., 1993. Mutational analysis of the Golgi retention signal of

bovine b-1,4-Galactosyltransferase. J. Biol. Chem. 268, 9908–9916.

Matlack, K.E., Mothes, W., Rapoport, T.A., 1998. Protein translocation: tunnel vision. Cell 92, 381–390.

Matter, K., Mellman, I., 1994. Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr. Opin. Cell

Biol. 6, 545–554.

Mayer, A., Wickner, W., Haas, A., 1996. Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking

and fusion of yeast vacuoles. Cell 85, 83–94.

McBride, H.M., Rybin, V., Murphy, C., Giner, A., Teasdale, R., Zerial, M., 1999. Oligomeric complexes link Rab5

effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13. Cell 98, 377–386.

McConville, M.J., Ilgoutz, S.C., Teasdale, R.D., Foth, B.J., Matthews, A., Mullin, K.A., Gleeson, P.A., 2002.

Targeting of the GRIP domain to the trans-Golgi network is conserved from protists to animals. Eur. J. Cell Biol.

81, 485–495.

McNew, J.A., Parlati, F., Fukuda, R., Johnston, R.J., Paz, K., Paumet, F., Sollner, T.H., Rothman, J.E., 2000a.

Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature 407, 153–159.

McNew, J.A., Weber, T., Parlati, F., Johnston, R.J., Melia, T.J., Sollner, T.H., Rothman, J.E., 2000b. Close is not

enough: snare-dependent membrane fusion requires an active mechanism that transduces force to membrane

anchors. J. Cell Biol. 150, 105–117.

Melkonian, M., Becker, B., Becker, D., 1991. Scale formation in algae. J. Electron. Microsc. Technol. 17, 15–23.

Mellman, I., 1996. Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575–625.

Meyer, C., Zizioli, D., Lausmann, S., Eskelinen, E.L., Hamann, J., Saftig, P., von Figura, K., Schu, P., 2000. Mu 1A-

adaptin-deficient mice: Lethality, loss of ap-1 binding and rerouting of mannose 6-phosphate receptors. EMBO J.

19, 2193–2203.

Milland, J., Russell, S.M., Dodson, H.C., McKenzie, I.F., Sandrin, M.S., 2002. The cytoplasmic tail of alpha 1,3-

galactosyltransferase inhibits Golgi localization of the full-length enzyme. J. Biol. Chem. 277, 10374–10378.

Mills, I.G., Jones, A.T., Clague, M.J., 1998. Involvement of the endosomal autoantigen EEA1 in homotypic fusion of

early endosomes. Curr. Biol. 8, 881–884.

Miranda, K.C., Khromykh, T., Christy, P., Le, T.L., Gottardi, C.J., Yap, A.S., Stow, J.L., Teasdale, R.D., 2001. A

dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1

epithelial cells. J. Biol. Chem. 276, 22565–22572.

Mironov, A.A., Beznoussenko, G.V., Nicoziani, P., Martella, O., Trucco, A., Kweon, H.S., Di Giandomenico, D.,

Polishchuk, R.S., Fusella, A., Lupetti, P., Berger, E.G., Geerts, W.J., Koster, A.J., Burger, K.N., Luini, A., 2001.


Small cargo proteins and large aggregates can traverse the Golgi by a common mechanism without leaving the

lumen of cisternae. J. Cell Biol. 155, 1225–1238.

Molloy, S.S., Thomas, L., VanSlyke, J.K., Stenberg, P.E., Thomas, G., 1994. Intracellular trafficking and activation of

the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J. 13, 18–33.

Moreau, P., Cassagne, C., 1994. Phospholipid trafficking and membrane biogenesis. Biochim. Biophys. Acta 1197,

257–290.

Morr!e, D.J., 1987. The Golgi apparatus. Int. Rev. Cytol. Suppl. 17, 211–253.

Mostov, K.E., Verges, M., Altschuler, Y., 2000. Membrane traffic in polarized epithelial cells. Curr. Opin. Cell Biol. 12,

483–490.

Moyer, B.D., Allan, B.B., Balch, W.E., 2001. Rab1 interaction with a GM130 effector complex regulates COPII vesicle

cis-Golgi tethering. Traffic 2, 268–276.

Mu, F.T., Callaghan, J.M., Steele-Mortimer, O., Stenmark, H., Parton, R.G., Campbell, P.L., McCluskey, J., Yeo,

J.P., Tock, E.P., Toh, B.H., 1995. EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical

peripheral membrane protein flanked by cysteine ‘‘fingers’’ and contains a calmodulin-binding IQ motif. J. Biol.

Chem. 270, 13503–13511.

Munro, S., 1991. Sequences within and adjacent to the transmembrane segment of alpha-2,6-sialyltransferase specify

Golgi retention. EMBO J. 10, 3577–3588.

Munro, S., 1995a. A comparison of the transmembrane domains of Golgi and plasma membrane proteins. Biochem.

Soc. Trans. 23, 527–530.

Munro, S., 1995b. An investigation of the role of transmembrane domains in Golgi protein retention. EMBO J. 14,

4695–4704.

Munro, S., 1998. Localisation of proteins to the Golgi apparatus. Trends Cell Biol. 8, 11–50615.

Munro, S., 2002. Organelle identity and the targeting of peripheral membrane proteins. Curr. Opin. Cell Biol. 14, 506.

Munro, S., Pelham, H.R., 1987. A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899–907.

Munro, S., Nichols, B.J., 1999. The GRIP domain—a novel Golgi-targeting domain found in several coiled-coil

proteins. Curr. Biol. 9, 377–380.

Nakamura, N., Lowe, M., Levine, T.P., Rabouille, C., Warren, G., 1997. The vesicle docking protein p115 binds

GM130, a cis-Golgi matrix protein, in mitotically regulated manner. Cell 89, 445–455.

Nakamura, N., Rabouille, C., Watson, R., Nilsson, T., Hui, N., Slusarewicz, P., Kreis, T.E., Warren, G., 1995.

Characterization of a cis-Golgi matrix protein, GM130. J. Cell Biol. 131, 1715–1726.

Nakano, A., Brada, D., Schekman, R., 1988. A membrane glycoprotein, Sec12p, required for protein transport from

the endoplasmic reticulum to the Golgi apparatus in yeast. J. Cell Biol. 107, 851–863.

Narula, N., Stow, J.L., 1995. Distinct coated vesicles labeled for p200 bud from trans-Golgi network membranes. Proc.

Natl. Acad. Sci. USA 92, 2874–2878.

Nelson, D.S., Alvarez, C., Gao, Y.S., Garcia-Mata, R., Fialkowski, E., Sztul, E., 1998. The membrane transport factor

TAP/p115 cycles between the Golgi and earlier secretory compartments and contains distinct domains required for

its localization and function. J. Cell Biol. 143, 319–331.

Nelson, W.J., Yeaman, C., 2001. Protein trafficking in the exocytic pathway of polarized epithelial cells. Trends Cell

Biol. 11, 483–486.

Neutra, M., Leblond, C.P., 1966. Synthesis of the carbohydrate of mucus in the Golgi complex as shown by electron

microscope radioautography of goblet cells from rats injected with glucose-H3. J. Cell Biol. 30, 119–136.

Nezil, F.A., Bloom, M., 1992. Combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer

thickness in model membranes. Biophys J. 61, 1176–1183.

Nichols, B.J., Pelham, H.R.B., 1998. SNAREs and membrane fusion in the Golgi apparatus. Biochim. Biophys. Acta

1404, 9–31.

Nielsen, E., Severin, F., Backer, J.M., Hyman, A.A., Zerial, M., 1999. Rab5 regulates motility of early endosomes on

microtubules. Nat. Cell Biol. 1, 376–382.

Nielsen, M.S., Madsen, P., Christensen, E.I., Nykjaer, A., Gliemann, J., Kasper, D., Pohlmann, R., Petersen, C.M.,

2001. The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2

sorting protein. EMBO J. 20, 2180–2190.


Nilsson, T., Slusarewicz, P., Warren, G., 1993. Kin recognition. A model for the retention of Golgi enzymes. FEBS 330,

1–4.

Nilsson, T., Hoe, M.H., Slusarewicz, P., Rabouille, C., Watson, R., Hunte, F., Watzele, G., Berger, E.G., Warren, G.,

1994. Kin Recognition Between medial Golgi Enzymes in HeLa Cells. EMBO J. 13, 562–574.

Nilsson, T., Rabouille, C., Hui, N., Watson, R., Warren, G., 1996. The role of the membrane-spanning domain and

stalk region of N-acetylglucosaminlytransferase I in retention, kin recognition and structural maintenance of the

Golgi apparatus in HeLa cells. J. Cell Sci. 109, 1975–1989.

Nishikawa, S., Nakano, A., 1993. Identification of a gene required for membrane protein retention in the early secretory

pathway. Proc. Natl. Acad. Sci. USA 90, 8179–8183.

Nishimura, N., Balch, W.E., 1997. A di-acidic signal required for selective export from the endoplasmic reticulum.

Science 277, 556–558.

Nishimura, N., Bannykh, S., Slabough, S., Matteson, J., Altschuler, Y., Hahn, K., Balch, W.E., 1999. A di-acidic

(DXE) code directs concentration of cargo during export from the endoplasmic reticulum. J. Biol. Chem. 274,

15937–15946.

Nothwehr, S.F., Hindes, A.E., 1997. The yeast VPS5/GRD2 gene encodes a sorting nexin-1-like protein required for

localizing membrane proteins to the late Golgi. J. Cell Sci. 110, 1063–1072.

Nothwehr, S.F., Ha, S.A., Bruinsma, P., 2000. Sorting of yeast membrane proteins into an endosome-to-Golgi pathway

involves direct interaction of their cytosolic domains with Vps35p. J. Cell Biol. 151, 297–310.

Novick, P., Ferro, S., Schekman, R., 1981. Order of events in the yeast secretory pathway. Cell 25, 461–469.

Novikoff, A.B., Goldfischer, S., 1961. Proc. Natl Acad Sci USA 47, 802–810.

Ohno, H., Stewart, J., Fournier, M.C., Bosshart, H., Rhee, I., Miyatake, S., Saito, T., Gallusser, A., Kirchhausen, T.,

Bonifacino, J.S., 1995. Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science 269,

1872–1875.

Olkkonen, V.M., Ikonen, E., 2000. Mechanisms of disease—genetic defects of intracellular-membrane transport. New

Eng. J. Med. 343, 1095–1104.

Opat, A.S., Houghton, F., Gleeson, P.A., 2000. Medial Golgi but not late Golgi glycosyltransferases exist as high

molecular weight complexes—role of luminal domain in complex formation and localization. J. Biol. Chem. 275,

11836–11845.

Opat, A.O., Houghton, F., Gleeson, P.A., 2001. Steady-state localisation of a medial-Golgi glycosyltransferase involves

transit through the trans-Golgi network. Biochem. J. 358, 33–40.

Orci, L., Glick, B.S., Rothman, J.E., 1986. A new type of coated vesicular carrier that appears not to contain clathrin:

its possible role in protein transport within the Golgi stack. Cell 46, 171–184.

Orci, L., Montesano, R., Meda, P., Malaisse-Lagae, F., Brown, D., Perrelet, A., Vassalli, P., 1981. Heterogeneous

distribution of filipin–cholesterol complexes across the cisternae of the Golgi apparatus. Proc. Natl. Acad. Sci. USA

78, 293–297.

Orci, L., Palmer, D.J., Ravazzola, M., Perrelet, A., Amherdt, M., Rothman, J.E., 1993. Budding from Golgi

membranes requires the coatomer complex of non-clathrin coat proteins. Nature 362, 648–652.

Orci, L., Perrelet, A., Rothman, J.E., 1998. Vesicles on strings: Morphological evidence for processive transport within

the Golgi stack. Proc. Natl. Acad. Sci. USA 95, 2279–2283.

Orci, L., Ravazzola, M., Volchuk, A., Engel, T., Gmachl, M., Amherdt, M., Perrelet, A., Sollner, T.H., Rothman, J.E.,

2000. Anterograde flow of cargo across the Golgi stack potentially mediated via bidirectional ‘‘percolating’’ COPI

vesicles. Proc. Natl. Acad. Sci. USA 97, 10400–10405.

Orci, L., Stamnes, M., Ravazzola, M., Amherdt, M., Perrelet, A., Sollner, T.H., Rothman, J.E., 1997. Bidirectional

transport by distinct populations of copi-coated vesicles. Cell 90, 335–349.

Palade, G., 1975. Intracellular Aspects of the Process of Protein Synthesis. Science 189, 347–358.

Parks, W.T., Frank, D.B., Huff, C., Renfrew Haft, C., Martin, J., Meng, X., de Caestecker, M.P., McNally, J.G.,

Reddi, A., Taylor, S.I., Roberts, A.B., Wang, T., Lechleider, R.J., 2001. Sorting nexin 6, a novel SNX, interacts

with the transforming growth factor-beta family of receptor serine-threonine kinases. J. Biol Chem. 276,

19332–19339.

Parlati, F., McNew, J.A., Fukuda, R., Miller, R., S .ollner, T.H., Rothman, J.E., 2000. Topological restriction of

SNARE-dependent membrane fusion. Nature 407, 194–198.


Parodi, A.J., 2000. Role of N-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and

degradation. Biochem. J. 348 (1), 1–13.

Paulson, J.C., Colley, K.J., 1989. Glycosyltransferases. Structure, localization, and control of cell type-specific

glycosylation. J. Biol. Chem. 264, 17615–17618.

Pelham, H.R., 2001. SNAREs and the specificity of membrane fusion. Trends Cell Biol. 11, 99–101.

Pelham, H.R.B., 1998. Getting through the Golgi complex. Trends Cell Biol. 8, 45–49.

Pelham, H.R., Hardwick, K.G., Lewis, M.J., 1988. Sorting of soluble ER proteins in yeast. EMBO J. 7, 1757–1762.

Pelham, H.R.B., Rothman, J.E., 2000. The debate about transport in the Golgi—two sides of the same coin? Cell 102,

713–719.

Pepperkok, R., Scheel, J., Horstmann, H., Hauri, H.P., Griffiths, G., Kreis, T.E., 1993. Beta-COP is essential for

biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo. Cell 74, 71–82.

Petris, M.J., Camakaris, J., Greenough, M., LaFontaine, S., Mercer, J.F., 1998. A C-terminal di-leucine is required for

localization of the Menkes protein in the trans-Golgi network. Hum. Mol. Genet. 7, 2063–2071.

Petris, M.J., Mercer, J.F.B., Culvenor, J.G., Lockhart, P., Gleeson, P.A., Camakaris, J., 1996. Ligand-regulated

transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a

novel mechanism of regulated trafficking. EMBO J. 15, 6084–6095.

Pevsner, J., Hsu, S.C., Braun, J.E., Calakos, N., Ting, A.E., Bennett, M.K., Scheller, R.H., 1994. Specificity and

regulation of a synaptic vesicle docking complex. Neuron 13, 353–361.

Pfeffer, S.R., 1999. Transport-vesicle targeting: tethers before snares. Nat. Cell Biol. 1, E17–22.

Pierre, P., Scheel, J., Rickard, J.E., Kreis, T.E., 1992. CLIP-170 links endocytic vesicles to microtubules. Cell 70,

887–900.

Pohlmann, R., Waheed, A., Hasilik, A., von Figura, K., 1982. Synthesis of phosphorylated recognition marker in

lysosomal enzymes is located in the cis part of Golgi apparatus. J. Biol. Chem. 257, 5323–5325.

Ponnambalam, S., Rabouille, C., Luzio, J.P., Nilsson, T., Warren, G., 1994. The TGN38 glycoprotein contains two

non-overlapping signals that mediate localization to the trans-Golgi network. J. Cell Biol. 125, 253–268.

Ponting, C.P., 1996. Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding

partners of SH3 domains? Protein Sci. 5, 2353–2357.

Poussu, A., Lohi, O., Lehto, V.P., 2000. Vear, a novel Golgi-associated protein with VHS and gamma-adaptin ‘‘ear’’

domains. J. Biol. Chem. 275, 7176–7183.

Presley, J.F., Cole, N.B., Schroer, T.A., Hirschberg, K., Zaal, K.J.M., Lippincottschwartz, J., 1997. ER-to-Golgi

transport visualized in living cells. Nature 389, 81–85.

Price, A., Wickner, W., Ungermann, C., 2000. Proteins needed for vesicle budding from the Golgi complex are also

required for the docking step of homotypic vacuole fusion. J. Cell Biol. 148, 1223–1229.

Puertollano, R., Aguilar, R.C., Gorshkova, I., Crouch, R.J., Bonifacino, J.S., 2001a. Sorting of mannose 6-phosphate

receptors mediated by the GGAs. Science 292, 1712–1716.

Puertollano, R., Randazzo, P.A., Presley, J.F., Hartnell, L.M., Bonifacino, J.S., 2001b. The GGAs promote ARF-

dependent recruitment of clathrin to the TGN. Cell 105, 93–102.

Rabouille, C., Hui, N., Hunte, F., Kieckbusch, R., Berger, E.G., Warren, G., Nilsson, T., 1995a. Mapping the

distribution of Golgi enzymes involved in the construction of complex oligosaccharides. J. Cell Sci. 108, 1617–1627.

Rabouille, C., Levine, T.P., Peters, J.M., Warren, G., 1995b. An NSF-like ATPase, p97, and NSF mediate cisternal

regrowth from mitotic Golgi fragments. Cell 82, 905–914.

Rapak, A., Falnes, P.O., Olsnes, S., 1997. Retrograde transport of mutant ricin to the endoplasmic reticulum with

subsequent translocation to cytosol. Proc. Natl. Acad. Sci. USA 94, 3783–3788.

Rapoport, T.A., Jungnickel, B., Kutay, U., 1996. Protein transport across the eukaryotic endoplasmic reticulum and

bacterial inner membranes. Annu. Rev. Biochem. 65, 271–303.

Raymond, C.K., Howald-Stevenson, I., Vater, C.A., Stevens, T.H., 1992a. Morphological classification of the yeast

vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol. Biol. Cell 3,

1389–1402.

Raymond, C.K., Roberts, C.J., Moore, K.E., Howald, I., Stevens, T.H., 1992b. Biogenesis of the vacuole in

Saccharomyces cerevisiae. Int. Rev. Cytol. 139, 59–120.


Rexach, M.F., Latterich, M., Schekman, R.W., 1994. Characteristics of endoplasmic reticulum-derived transport

vesicles. J. Cell Biol. 126, 1133–1148.

Rios, R.M., Tassin, A.M., Celati, C., Antony, C., Boissier, M.C., Homberg, J.C., Bornens, M., 1994. A peripheral

protein associated with the cis-Golgi network redistributes in the intermediate compartment upon brefeldin A

treatment. J. Cell Biol. 125, 997–1013.

Robinson, M.S., Bonifacino, J.S., 2001. Adaptor-related proteins. Curr. Opin. Cell Biol. 13, 444–453.

Robinson, J.S., Klionsky, D.J., Banta, L.M., Emr, S.D., 1988. Protein sorting in Saccharomyces cerevisiae: isolation

of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol. Cell Biol. 8,

4936–4948.

Rodman, J.S., Wandinger-Ness, A., 2000. Rab GTPases coordinate endocytosis—commentary. J. Cell Sci. 113,

183–192.

Rohn, W.M., Rouille, Y., Waguri, S., Hoflack, B., 2000. Bi-directional trafficking between the trans-Golgi network and

the endosomal/lysosomal system. J. Cell Sci. 113, 2093–2101.

Roquemore, E.P., Banting, G., 1998. Efficient trafficking of TGN38 from the endosome to the trans-Golgi network

requires a free hydroxyl group at position 331 in the cytosolic domain. Mol. Biol. Cell 9, 2125–2144.

Roth, J., Berger, E.G., 1982. Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution

with thiamine pyrophosphatase in trans-Golgi cisternae. J. Cell Biol. 93, 223–229.

Rothman, J.E., 1994. Mechanism of intracellular protein transport. Nature 372, 55–63.

Rothman, J.E., Orci, L., 1992. Molecular dissection of the secretory pathway. Nature 355, 409–415.

Rothman, J.E., Wieland, F.T., 1996. Protein sorting by transport vesicles. Science 272, 227–234.

Rothman, J.H., Stevens, T.H., 1986. Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize

vacuolar proteins into the late secretory pathway. Cell 47, 1041–1051.

Rouille, Y., Rohn, W., Hoflack, B., 2000. Targeting of lysosomal proteins. Semin. Cell Dev. Biol. 11, 165–171.

Sapperstein, S.K., Lupashin, V.V., Schmitt, H.D., Waters, M.G., 1996. Assembly of the ER to Golgi SNARE complex

requires Uso1p. J. Cell Biol. 132, 755–767.

Saraste, J., Kuismanen, E., 1984. Pre- and post-Golgi vacuoles operate in the transport of Semliki Forest virus

membrane glycoproteins to the cell surface. Cell 38, 535–549.

Saraste, J., Kuismanen, E., 1992. Pathways of protein sorting and membrane traffic between the rough endoplasmic

reticulum and the Golgi complex. Semin. Cell Biol. 3, 343–355.

Sasai, K., Ikeda, Y., Tsuda, T., Ihara, H., Korekane, H., Shiota, K., Taniguchi, N., 2001. The critical role of the stem

region as a functional domain responsible for the oligomerization and Golgi localization of N-acetylglucosaminyl-

transferase V. The involvement of a domain homophilic interaction. J. Biol. Chem. 276, 759–765.

Sato, K., Nishikawa, S., Nakano, A., 1995. Membrane protein retrieval from the Golgi apparatus to the endoplasmic

reticulum (ER): characterization of the RER1 gene product as a component involved in ER localization of Sec12p.

Mol. Biol. Cell 6, 1459–1477.

Sato, M., Sato, K., Nakano, A., 1996. Endoplasmic reticulum localization of Sec12p is achieved by two mechanisms:

Rer1p-dependent retrieval that requires the transmembrane domain and Rer1p-independent retention that involves

the cytoplasmic domain. J. Cell Biol. 134, 279–293.

Scales, S.J., Pepperkok, R., Kreis, T.E., 1997. Visualization of ER-to-Golgi transport in living cells reveals a sequential

mode of action for COPII and COPI. Cell 90, 1137–1148.

Schachter, H., Narasimhan, S., Gleeson, P., Vella, G., 1983. Control of branching during the biosynthesis of

asparagine-linked oligosaccharides. Can. J. Biochem. Cell Biol. 61, 1049–1066.

Schekman, R., Orci, L., 1996. Coat proteins and vesicle budding. Science 271, 1526–1533.

Schmid, S., 1997. Clathrin-coated vesicle formation and protein sorting: an integrated process. Annu. Rev. Biochem.

66, 511–548.

Schroer, T.A., 2000. Motors, clutches and brakes for membrane traffic: a commemorative review in honor of Thomas

Kreis. Traffic 1, 3–10.

Schultz, J., Copley, R.R., Doerks, T., Ponting, C.P., Bork, P., 2000. SMART: A web-based tool for the study of

genetically mobile domains. Nucleic Acids Res. 28, 231–234.

Schutze, M.P., Peterson, P.A., Jackson, M.R., 1994. An N-terminal double-arginine motif maintains type II membrane

proteins in the endoplasmic reticulum. EMBO J. 13, 1696–1705.


Schweizer, A., Fransen, J.A., Matter, K., Kreis, T.E., Ginsel, L., Hauri, H.P., 1990. Identification of an intermediate

compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus. Eur. J. Cell Biol. 53,

185–196.

Seaman, M.N., Marcusson, E.G., Cereghino, J.L., Emr, S.D., 1997. Endosome to Golgi retrieval of the vacuolar

protein sorting receptor, Vps10p, requires the function of the VPS29, VPS30, and VPS35 gene products. J. Cell Biol.

137, 79–92.

Seaman, M.N., McCaffery, J.M., Emr, S.D., 1998. A membrane coat complex essential for endosome-to-Golgi

retrograde transport in yeast. J. Cell Biol. 142, 665–681.

Seemann, J., Jokitalo, E.J., Warren, G., 2000. The role of the tethering proteins p115 and GM130 in transport through

the Golgi apparatus in vivo. Mol. Biol. Cell 11, 635–645.

Segev, N., 2001. Ypt and Rab GTPases: insight into functions through novel interactions. Curr. Opin. Cell Biol. 13,

500–511.

Semenza, J.C., Hardwick, K.G., Dean, N., Pelham, H.R., 1990. ERD2, a yeast gene required for the receptor-mediated

retrieval of luminal ER proteins from the secretory pathway. Cell 61, 1349–1357.

Shorter, J., Warren, G., 1999. A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling

Golgi cisternae in a cell-free system. J. Cell Biol. 146, 57–70.

Simons, K., Ikonen, E., 1997. Functional rafts in cell membranes. Nature 387, 569–572.

Simonsen, A., Lippe, R., Christoforidis, S., Gaullier, J.M., Brech, A., Callaghan, J., Toh, B.H., Murphy, C., Zerial, M.,

Stenmark, H., 1998. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature 394,

494–498.

Simonsen, A., Gaullier, J.M., D’Arrigo, A., Stenmark, H., 1999. The Rab5 effector EEA1 interacts directly with

syntaxin-6. J. Biol. Chem. 274, 28857–28860.

Sj .ostrand, F.S., Hanzon, V., 1954. Exp. Cell Res. 7, 425–429.

S .ollner, T., Bennett, M.K., Whiteheart, S.W., Scheller, R.H., Rothman, J.E., 1993a. A protein assembly disassembly

pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75,

409–418.

S .ollner, T., Whiteheart, S., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., Rothman, J., 1993b.

SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324.

S .onnichsen, B., Fullekrug, J., Nguyen Van, P., Diekmann, W., Robinson, D.G., Mieskes, G., 1994. Retention and

retrieval: both mechanisms cooperate to maintain calreticulin in the endoplasmic reticulum. J. Cell Sci. 107,

2705–2717.

Sonnichsen, B., Lowe, M., Levine, T., Jamsa, E., Dirac-Svejstrup, B., Warren, G., 1998. A role for giantin in docking

COPI vesicles to Golgi membranes. J. Cell Biol. 140, 1013–1021.

Spang, A., Matsuoka, K., Hamamoto, S., Schekman, R., Orci, L., 1998. Coatomer, Arf1p, and nucleotide are

required to bud coat protein complex I-coated vesicles from large synthetic liposomes. Proc. Natl. Acad. Sci. USA

95, 11199–11204.

Springer, S., Schekman, R., 1998. Nucleation of COPII vesicular coat complex by endoplasmic reticulum to Golgi

vesicle SNAREs. Science 281, 698–700.

Springer, S., Chen, E., Duden, R., Marzioch, M., Rowley, A., Hamamoto, S., Merchant, S., Schekman, R., 2000. The

p24 proteins are not essential for vesicular transport in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 97,

4034–4039.

Stamnes, M.A., Craighead, M.W., Hoe, M.H., Lampen, N., Geromanos, S., Tempst, P., Rothman, J.E., 1995. An

integral membrane component of coatomer-coated transport vesicles defines a family of proteins involved in

budding. Proc. Natl. Acad. Sci. USA 92, 8011–8015.

Stenmark, H., Aasland, R., Toh, B.H., D’Arrigo, A., 1996. Endosomal localization of the autoantigen EEA1 is

mediated by a zinc-binding FYVE finger. J. Biol. Chem. 271, 24048–24054.

Stephens, D.J., Pepperkok, R., 2001. Illuminating the secretory pathway: When do we need vesicles? J. Cell Sci. 114,

1053–1059.

Stephens, D.J., Crump, C.M., Clarke, A.R., Banting, G., 1997. Serine 331 and tyrosine 333 are both involved in the

interaction between the cytosolic domain of TGN38 and the mu2 subunit of the AP2 clathrin adaptor complex.

J. Biol. Chem. 272, 14104–14109.


Sweitzer, S.M., Hinshaw, J.E., 1998. Dynamin undergoes a GTP-dependent conformational change causing

vesiculation. Cell 93, 1021–1029.

Tai, A.W., Chuang, J.Z., Bode, C., Wolfrum, U., Sung, C.H., 1999. Rhodopsin’s carboxy-terminal cytoplasmic tail

acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1. Cell 97,

877–887.

Takahashi, S., Nakagawa, T., Banno, T., Watanabe, T., Murakami, K., Nakayama, K., 1995. Localization of furin to

the trans-golgi network and recycling from the cell surface involves Ser and Tyr residues within the cytoplasmic

domain. J. Biol. Chem. 270, 28397–28401.

Tang, B.L., Wong, S.H., Low, S.H., Hong, W., 1992. The transmembrane domain of N-glucosaminyltransferase I

contains a Golgi retention signal. J. Biol. Chem. 267, 10122–10126.

Teasdale, R.D., Jackson, M.R., 1996. Signal-mediated sorting of membrane proteins between the endoplasmic

reticulum and the Golgi apparatus. Annu. Rev. Cell Dev. Biol. 12, 27–54.

Teasdale, R.D., Loci, D., Houghton, F., Karlsson, L., Gleeson, P.A., 2001. A large family of endosome-localized

proteins related to sorting nexin 1. Biochem. J. 358, 7–16.

TerBush, D.R., Maurice, T., Roth, D., Novick, P., 1996. The Exocyst is a multiprotein complex required for exocytosis

in Saccharomyces cerevisiae. EMBO J. 15, 6483–6494.

Thiele, C., Gerdes, H.H., Huttner, W.B., 1997. Protein secretion: Puzzling receptors. Curr. Biol. 7, R496–500.

Thyberg, J., Moskalewski, S., 1999. Role of microtubules in the organization of the Golgi complex. Exp. Cell Res. 246,

263–279.

Toomre, D., Keller, P., White, J., Olivo, J.C., Simons, K., 1999. Dual-color visualization of trans-Golgi network to

plasma membrane traffic along microtubules in living cells. J. Cell Sci. 112, 21–33.

Traub, L.M., Kornfeld, S., 1997. The trans-Golgi network—a late secretory sorting station. Curr. Opin. Cell Biol. 9,

527–533.

Trombetta, S.E., Parodi, A.J., 1992. Purification to apparent homogeneity and partial characterization of rat liver

UDP-glucose: glycoprotein glucosyltransferase. J. Biol. Chem. 267, 9236–9240.

Tsui, M.M.K., Banfield, D.K., 2000. Yeast Golgi SNARE interactions are promiscuous. J. Cell Sci. 113, 145–152.

Ungermann, C., Nichols, B., Pelham, H., Wickner, W., 1998. A vacuolar v-t-SNARE complex, the predominant form

in vivo and on isolated vacuoles and on isolated vacuoles, is disassembled and activated for docking and fusion.

J. Cell Biol. 140, 61–69.

Ungewickell, E., Ungewickell, H., Holstein, S.E., Lindner, R., Prasad, K., Barouch, W., Martin, B., Greene, L.E.,

Eisenberg, E., 1995. Role of auxilin in uncoating clathrin-coated vesicles. Nature 378, 632–635.

van der Bliek, A.M., Redelmeier, T.E., Damke, H., Tisdale, E.J., Meyerowitz, E.M., Schmid, S.L., 1993. Mutations in

human dynamin block an intermediate stage in coated vesicle formation. J. Cell Biol. 122, 553–563.

van Meer, G., 1998. Lipids of the Golgi membrane. Trends Cell Biol. 8, 29–33.

Varki, A., 1998. Factors controlling the glycosylation potential of the Golgi apparatus. Trends Cell Biol. 8, 34–40.

von Mollard, G.F., Nothwehr, S.F., Stevens, T.H., 1997. The yeast v-SNARE Vti1p mediates two vesicle transport

pathways through interactions with the t-SNAREs Sed5p and Pep12p. J. Cell Biol. 137, 1511–1524.

Walter, P., Johnson, A.E., 1994. Signal sequence recognition and protein targeting to the endoplasmic reticulum

membrane. Annu. Rev. Cell Biol. 10, 87–119.

Wan, L., Molloy, S.S., Thomas, L., Liu, G., Xiang, Y., Rybak, S.L., Thomas, G., 1998. PACS-1 defines a novel gene

family of cytosolic sorting proteins required for trans-Golgi network localization. Cell 94, 205–216.

Warren, G., Mellman, I., 1999. Bulk flow redux? Cell 98, 125–127.

Waters, M.G., Hughson, F.M., 2000. Membrane tethering and fusion in the secretory and endocytic pathways. Traffic

1, 588–597.

Waters, M.G., Pfeffer, S.R., 1999. Membrane tethering in intracellular transport. Curr. Opin. Cell Biol. 11, 453–459.

Waters, M.G., Clary, D.O., Rothman, J.E., 1992. A novel 115-kD peripheral membrane protein is required for

intercisternal transport in the Golgi stack. J. Cell Biol. 118, 1015–1026.

Waters, M.G., Serafini, T., Rothman, J.E., 1991. ‘Coatomer’: a cytosolic protein complex containing subunits of non-

clathrin-coated Golgi transport vesicles. Nature 349, 248–251.

Watts, C., 1997. Capture and processing of exogenous antigens for presentation on MHC molecules. Annu. Rev.

Immunol. 15, 821–850.


Weber, T., Zemelman, B., McNew, J., Westermann, B., Gmachl, M., Parlati, F., Sollner, T., Rothman, J., 1998.

SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772.

Wieland, F.T., Gleason, M.L., Serafini, T.A., Rothman, J.E., 1987. The rate of bulk flow from the endoplasmic

reticulum to the cell surface. Cell 50, 289–300.

Wilson, D.W., Lewis, M.J., Pelham, H.R., 1993. pH-dependent binding of KDEL to its receptor in vitro. J. Biol. Chem.

268, 7465–7468.

Wishart, M.J., Taylor, G.S., Dixon, J.E., 2001. Phoxy lipids: revealing PX domains as phosphoinositide binding

modules. Cell 105, 817–820.

Wong, S.H., Hong, W.J., 1993. The SXYQRL sequence in the cytoplasmic domain of TGN38 plays a major role in

trans-Golgi network localization. J. Biol. Chem. 268, 22853–22862.

Worby, C.A., Dixon, J.E., 2002. Sorting out the cellular functions of sorting nexins. Nature Rev. Mol. Cell Biol. 3,

919–931.

Wurmser, A.E., Sato, T.K., Emr, S.D., 2000. New component of the vacuolar class C-Vps complex couples nucleotide

exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion. J. Cell Biol. 151, 551–562.

Xu, Y., Hortsman, H., Seet, L., Wong, S.H., Hong, W., 2001. SNX3 regulates endosomal function through its PX-

domain-mediated interaction with PtdIns(3)P. Nat. Cell Biol. 3, 658–666.

Yeaman, C., Grindstaff, K.K., Wright, J.R., Nelson, W.J., 2001. Sec6/8 complexes on trans-Golgi network and plasma

membrane regulate late stages of exocytosis in mammalian cells. J. Cell Biol. 155, 593–604.

Yeung, T., Barlowe, C., Schekman, R., 1995. Uncoupled packaging of targeting and cargo molecules during transport

vesicle budding from the endoplasmic reticulum. J. Biol. Chem. 270, 30567–30570.

Yoshihisa, T., Barlowe, C., Schekman, R., 1993. Requirement for a GTPase-activating protein in vesicle budding from

the endoplasmic reticulum. Science 259, 1466–1468.

Yuan, Z., Teasdale, R.D., 2002. Prediction of Golgi type II membrane proteins based on their transmembrane domains.

Bioinformatics 18, 1109–1115.

Zhao, L., Helms, J.B., Brugger, B., Harter, C., Martoglio, B., Graf, R., Brunner, J., Wieland, F.T., 1997. Direct and

GTP-dependent interaction of ADP ribosylation factor 1 with coatomer subunit beta. Proc. Natl. Acad. Sci. USA

94, 4418–4423.

Zhong, Q., Lazar, C.S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S.D., Gill, G.N., 2002.

Endosomal localization and function of sorting nexin 1. Proc. Natl. Acad. Sci. USA 99, 6767–6772.

Zhu, Y., Doray, B., Poussu, A., Lehto, V.P., Kornfeld, S., 2001. Binding of GGA2 to the lysosomal enzyme sorting

motif of the mannose 6-phosphate receptor. Science 292, 1716–1718.


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