Protein Folding before and after Translocation into the YeastEndoplasmic Reticulum

Eija Paunola

Institute of Biotechnology,Department of Biosciences, Division of Biochemistry,

andGraduate School in Microbiology and

Viikki Graduate School in Biosciences,Faculty of Science

University of HelsinkiFinland

Academic dissertation

To be presented, with the permission of the Faculty of Science of the University ofHelsinki, for public criticism in the auditorium 1041 at Viikki Biocenter (Viikinkaari 5,

Helsinki) on August 11 th, 2000, at 12 o’clock.

Helsinki 2000

Supervised by

Professor Marja MakarowInstitute of BiotechnologyUniversity of Helsinki

Reviewed by

Docent Pekka LappalainenInstitute of BiotechnologyUniversity of Helsinki

Doctor Vesa KontinenDepartment of VaccinesNational Public Health InstituteHelsinki

Opponent

Docent Kalervo MetsikköDepartment of AnatomyUniversity of Oulu

Kustos

Professor Carl G. GahmbergDepartment of BiosciencesDivision of BiochemistryUniversity of Helsinki

ISBN 951-45-9454-1 (nid)ISBN 952-91-2284-5 (verkkojulkaisu, pdf)ISSN 1239-9469

YliopistopainoHelsinki 2000

ORIGINAL PUBLICATIONS

This thesis is based on the following articles and a manuscript, which are referred to in thetext by their Roman numerals.

I Paunola E., Suntio T., Jämsä E. and Makarow M. 1998. Folding of active

β-lactamase in the yeast cytoplasm before translocation into theendoplasmic reticulum. Mol. Biol. Cell 9: 817-827.

II Paunola E., Qiao M. and Makarow M. Prefolded β-lactamase is unfolded

in vivo before translocation into the yeast ER. (submitted)

III Holkeri H., Paunola E., Jämsä E. and Makarow M. 1998. Dissection of thetranslocation and chaperoning functions of yeast BiP/Kar2p in vivo. J. CellSci. 111: 749-757.

CONTENTS

SUMMARY ............................................................................................................ 1

INTRODUCTION ................................................................................................... 2

1. Overview .......................................................................................................... 2

2. Protein translocation ........................................................................................ 3

2.1. Protein targeting to the endoplasmic reticulum ............................................. 32.1.1. Signal peptides ............................................................................................. 32.1.2. Cotranslational translocation .......................................................................... 32.1.3. Posttranslational translocation ........................................................................ 52.2. The translocation channel ............................................................................. 52.3. Chaperones in translocation .......................................................................... 92.4. Retrotranslocation ....................................................................................... 10

3. Protein maturation in the ER ........................................................................... 11

3.1. Glycosylation ............................................................................................... 123.1.1. N-glycosylation ........................................................................................... 123.1.2. O-glycosylation ........................................................................................... 123.2. Lectin-like chaperones: Calnexin and calreticulin ........................................ 133.3. Classical chaperones................................................................................... 143.4. Folding enzymes: Protein disulfide isomerases and peptidyl prolyl

isomerases................................................................................................... 15

AIMS OF THE STUDY ......................................................................................... 17

MATERIALS AND METHODS .............................................................................. 18

RESULTS ............................................................................................................ 21

1. The reporter proteins: Hsp150 and the fusion proteins................................... 21

2. Protein folding before translocation into the ER ( I ) ....................................... 22

2.1. Hsp150D is translocated posttranslationally (I) ............................................ 222.2. The translocation intermediate on the cytoplasmic face of the ER membrane

(I) .................................................................................................................. 222.3. The translocation intermediate is active (I) .................................................. 23

3. The conformation of proteins attached to the translocon (II) .......................... 23

3.1. The b-lactamase mutants (II) ........................................................................ 233.2. The effect of PenG on the reporter (II) .......................................................... 243.3. Subcellular localization of PenG-bound reporter protein (II) ........................ 243.4. The effect of reversibly bound drug on translocation (II) ............................. 253.5. An unfolding step prior to translocation (I, II)............................................... 25

4. Protein folding in the ER (III) ........................................................................... 26

4.1. The effect of DTT on secretion (III) ............................................................... 264.2. The role of BiP in conformational maturation and translocation (III) ............ 28

DISCUSSION....................................................................................................... 29

1. Pretranslocational protein folding................................................................... 29

2. Unfolding precedes translocation ................................................................... 30

3. The functions of BiP in translocation and folding are distinct ........................ 33

CONCLUSIONS ................................................................................................... 34

ACKNOWLEDGEMENTS ..................................................................................... 35

REFERENCES..................................................................................................... 36

ABBREVIATIONS

A alanineATP adenosine triphosphateBiP immunoglobulin heavy chain-binding proteinCHX cycloheximideCPY carboxy peptidase YD aspartic acidDTT dithithreitolE glutamic acidER endoplasmic reticulumGDP guanosine diphosphateGTP guanosine triphosphateHsp heat shock proteinKDa kilodaltonMHC major histocompatibility complexNAC nascent-chain associated complexNGFRe nerve growth factor receptor ectodomainPAGE polyacrylamide gel electrophoresisPDI protein disulfide isomerasePPI peptidyl-prolyl cis/trans isomeraseRAMP4 ribosome-associated membrane protein 4SDS sodium dodecyl sulphateSRP signal recognition particleTRAM translocating chain-associating proteinUb ubiquitinUGGT UDP-glucose:glycoprotein glucosyltransferaseUPRE unfolded protein response element

1

SUMMARY

Translocation of secretory proteins to theendoplasmic reticulum (ER) is the first stepin the secretory pathway. Artificial reporterproteins were used to studyposttranslational translocation and foldingin Saccharomyces cerevisiae cells. E. coliβ-lactamase and rat nerve growth factorreceptor ectodomain (NGFRe) were fusedto the Hsp150∆-carrier, an N-terminalfragment of a yeast secretory glycoprotein,whose signal peptide was found to conferposttranslational translocation. β-lactamasemutants binding to penicillin G in anirreversible or reversible fashion werecreated and fused to the carrier. β-lactamase is a tight globular protein andNGFRe consists of four separate extendeddomains. All fusion proteins were found tobe translocated posttranslationally andproperly folded in the yeast ER andthereafter secreted to the culture medium.

The β-lactamase portion of the fusionprotein was found to adopt a native-likeconformation in the yeast cytosol before itwas translocated into the ER. Similar to theauthentic protein, it was trypsin-resistant.Furthermore, it had similar catalytic activityand Km values for nitrocefin on both sidesof the ER membrane. The foldedcytoplasmic form of the fusion protein couldbe chased to the ER and to the culturemedium in active conformation. In contrastto the requirement of a disulfide bond foractivity in the ER, productive folding in the

cytoplasm was not dependent on disulfidebond formation, highlighting the differencesof the cytosol and the ER lumen as foldingmilieus. Translocation of the mutant β-lactamase fusion protein was found to beprevented when it was locked to a stableconformation due to the irreversibly boundpenicillin G. The prefolded β-lactamasefusion protein was attached to thetranslocation sites and unfolded beforeinsertion into the translocation pore wasinitiated. The results of this study show forthe first time that a polypeptide is folded inthe yeast cytoplasm before translocationinto the ER. They reveal a novel functionin the cytoplasm, unfolding of prefoldedprotein, resulting in signalling for poreopening.

In the ER lumen the chaperone BiP assistsin translocation and folding of newlysynthesised polypeptides. The fusionproteins Hsp150∆-β-lactamase andHsp150∆-NGFRe were used to study ifthese two functions were independent.Translocation of Hsp150∆-β-lactamaseand Hsp150∆-NGFRe were dependent onfunctional BiP. However, only the β-lactamase portion required BiP forconformational maturation in the ER. Thus,the requirement for BiP for folding isapparently substrate-specific, and someproteins, such as NGFRe, do not need tobe assisted by BiP.

2

INTRODUCTION

1. Overview

The eukaryotic cell contains membrane-bound organelles that perform highlyspecialized functions. Each organelle hasspecific proteins that define its structureand function. The maintenance of thesecompartments requires that newlysynthesized proteins are accuratelytargeted to their final destination.

All polypeptides destined for transportthrough the secretory pathway begin theirjourney by crossing the endoplasmicreticulum (ER) membrane. Aftertranslocation into the ER, proteins aretransported to the Golgi apparatus and fromthere to the lysosome or the vacuole inyeast, the plasma membrane, or theexterior of the cell, unless they are meant

to stay in the ER or Golgi. All transport stepsafter translocation into the ER occur byvesicular budding and fusion, andtranslocation across membranes is nolonger required. The secretory pathway ofyeast Saccharomyces cerevisiae has beendefined by temperature-sensitive secretionmutants, known as sec mutants, in whichthe transport of proteins is reversiblyblocked into dif ferent organelles of thepathway (Figure 1).

In the special folding environment of theER, proteins are modified co- andposttranslationally, and acquire their maturetertiary and quaternary structures. Eventhough the information needed for properfolding resides in the amino acid sequence

of the polypeptides, many proteinsassist and accelerate folding. If propermaturation fails, the aberrant productsare retained in the ER and eventuallydegraded. Thus, in addition to beinga special folding milieu, secretoryproteins are sorted in the ER fromresident proteins and incorrectlyfolded proteins, which if secretedcould be harmful or even dangerousto the cell.

Figure 1. A schematic picture of thesecretory pathway of S. cerevisiaepresenting the Sec proteins mentioned inthis study.

3

2. Protein translocation

2.1. Protein targeting to theendoplasmic reticulum

2.1.1. Signal peptides

In eukaryotic cells, most proteins aresynthesized on free ribosomes in thecytoplasm. They find their destination byspecific targeting sequences or signalpeptides. Signal peptides of secretoryproteins are usually N-terminal extensions(Blobel and Dobberstein, 1975), but canalso be located within a protein or at its C-terminal end (von Heijne, 1990).

Signal peptides vary in their amino acidcomposition and length, but they all containa positively charged N-terminus of one tofive residues, a central hydrophobic coreof six to fifteen amino acid residues and apolar region of three to seven amino acids.Studies with signal peptide mutantsrevealed that the hydrophobic core regionis the most essential part required fortargeting (von Heijne, 1985). The polarregion contains a recognition site for signalpeptidases (von Heijne, 1990), which areenzymes known to cut the signal peptide,once its targeting function has beencompleted. The cleavage occurs eitherduring translocation or soon aftercompletion of translocation. The yeastsignal peptidase is a heterotetramericprotein complex whose enzymatic activityis provided by the subunits Sec11p (Böhniet al., 1988; YaDeau et al., 1991) and Spc3p(Fang et al., 1997; Meyer and Hartmann,1997). In bacteria, a similar translocationsystem resides in the plasma membrane(Hartmann et al. , 1994). The crystalstructure of the bacterial signal peptidasehas been resolved (Paetzel et al., 1998).The active site of the peptidase issurrounded by an extended hydrophobic

patch suggested to position the active sitenear the membrane, where it meets thecleavage site of the translocating substrate.In the case of membrane proteintranslocation, the signal peptide can anchorthe protein in the membrane, instead ofbeing cleaved off (High and Dobberstein,1992).

Signal peptides can direct proteins to theER membrane through dif ferent targetingpathways. In yeast, translocation into theER can occur either during proteinsynthesis, i.e. cotranslationally, or afterprotein synthesis and release from theribosome, i.e. posttranslationally. Thehydrophobicity of the signal peptidedetermines which translocation pathway isused (Ng et al., 1996). Signal peptidesinteract with many proteins duringtranslocation, indicating that they are notmerely lipophilic peptides, but play an activerole during protein translocation into the ER.Mothes et al. (1998) reported that all stepsof cotranslational translocation, includingthe recognition of signal peptide, can bereproduced with purified translocationcomponents in detergent solution in theabsence of lipids, indicating that signalpeptides are ultimately recognized byprotein-protein interactions.

2.1.2. Cotranslational translocation

As secretory proteins emerge from theribosome during translation, the signalrecognition particle (SRP), a complex of sixpolypeptides (72, 68, 54, 19, 14, 9 kD) andan RNA component, binds to the signalpeptide (Walter et al., 1981). In addition toits ability to bind to signal peptides, SRPwas shown to cause a site-specific arrestin chain elongation that was released by

Introduction

4

binding to microsomal membranes (Walterand Blobel, 1981; Wolin and Walter, 1989).The arrest of protein synthesis gives timefor the nascent-chain ribosome complex tofind a binding site in the ER membranebefore domains of the polypeptide areexposed in the cytosol, which could lead totheir folding, misfolding or aggregation.

Analysis of the protein components of SRPhas identified subcomplexes responsiblefor the above-mentioned functions. SRP54protein binds to the signal sequence (Krieget al., 1986; Kurzchalia et al., 1986). It hasa methionine-rich domain, which isresponsible for the binding of signal peptide(High and Dobberstein, 1991). Zheng andNicchitta (1999) showed that a leucine-richsignal peptide is necessary for optimalinteraction with SRP and proposed thatSRP maintains the signal peptide in aconformation competent for binding to themembrane.The crystal structure of thesignal peptide binding subunit of the SRPfrom Thermus aquaticus showes a deepgroove bounded by a flexible loop and linedwith hydrophobic residues (Keenan et al.,1998). As a flexible, hydrophobicenvironment, the groove canaccommodate signal peptides of differentlengths and primary structures. Matoba andOgrydziak (1998) reported that factorsother than hydrophobicity, such asconformation or orientation of the signalpeptide, can affect its interaction with SRP.The elongation arrest is caused by theSRP9 and SRP14 subcomplexes (Siegeland Walter, 1985), and targeting to the ERmembrane requires SRP68 and SRP72subunits (Siegel and Walter, 1988).

The nascent chain-ribosome complex istargeted to the ER translocation machineryvia the interaction of SRP with the SRPreceptor in the ER membrane (Gilmore etal., 1982; Meyer et al., 1982). Binding ofGTP to SRP54 and to the SRP receptor

stabilizes the SRP-SRP receptor complex(Bacher et al., 1996; Rapiejko and Gilmore,1997) and leads to dissociation of SRPfrom the nascent chain and the ribosome(Connolly and Gilmore, 1989). Hydrolysisof GTP releases the SRP from the receptor(Connolly et al., 1991) and prepares it forthe next targeting round.

Homologues of mammalian SRPcomponents have been identified in yeast.Yeast Srp54p, a homologue of mammalianSRP54 protein, was found to be in acomplex with a major yeast cytoplasmicRNA, scR1, and to be important fortargeting of proteins to the ER (Hann andWalter, 1991). Stirling et al. (1992) isolateda temperature-sensitive yeast mutant,sec65-1, as being defective in membraneprotein insertion. Yeast Sec65p is ahomologue of SRP19 (Stirling and Hewitt,1992), and it was shown to be required forthe stable association of Srp54p with SRPand for the recycling of Srp54p (Hann etal., 1992; Regnacq et al., 1998). Brown etal. (1994) purified the yeast SRP and foundthat it contains homologues of mammalianSRP14, SRP68 and SRP72. Lack of anyof the SRP components led to slow cellgrowth and inefficient protein translocationinto the ER. The yeast SRP contains aprotein component, Srp21p, which appearsto have no counterpart in mammalian SRP.

The fidelity of the cotranslational targetingof a polypeptide is thought to be maintainedby the nascent polypeptide-associatedcomplex (NAC). NAC is an abundantheterodimeric protein complex thatinteracts with both cytoplasmic andsecretory nascent chains as they emergefrom the ribosome (Wiedman et al., 1994).In the absence of NAC, any ribosome wasable to bind to the ER membrane and evencytoplasmic proteins without signalpeptides could be translocated across themembrane. Thus, it was proposed that

5

NAC may be a negative regulator for ERtargeting. However, Neuhof et al. (1998)reported that SRP-independent targeting ofthe ribosome to the ER membrane occursin the absence or presence of NAC,indicating that NAC may have another role.In the absence of SRP, binding of ribosome-nascent chain complex to the ERmembrane was dependent on free Sec61p,which is a component of the translocationchannel, and nontranslating ribosomeswere found to compete for this interaction.In the presence of SRP, this competitionwas not observed. Thus, the binding ofSRP to ribosome-nascent chain complexesgives a competitive advantage in theinteraction with ER membrane. Signalpeptide-bound SRP has been shown tooccupy the membrane attachment site onribosomes and is therefore thought to inhibitthe binding of NAC (Möller et al., 1998).

2.1.3. Posttranslational translocation

In mammalian cells, most proteins aretranslocated cotranslationally, but shortpeptides and some proteins are able totranslocate posttranslationally without SRP(Schlenstedt et al., 1990). In yeast, a largernumber of proteins are translocatedposttranslationally. As a fast-growingorganism, its translation rate may exceedthe translocation rate (Matlack et al., 1998).Yeast cells are viable in the absence of SRP(Hann and Walter, 1991; Brown et al .,1994). Furthermore, cells lacking SRPadapt over time and thereby gain the abilityto translocate proteins, which aretranslocated cotranslationally undernormal conditions (Ogg et al., 1992). Thetime dependence of adaptation suggeststhat synthesis of specific proteins may berequired. Indeed, upon the depletion ofSRP54, the cytoplasmic levels of bothcytoplasmic Ssa1p chaperone and Ydj1p

cochaperone were increased (Arnold andWittrup, 1994), although this could also bea general stress response caused by theaccumulation of secretory proteinprecursors in the cytoplasm.

The targeting step of posttranslationaltranslocation is apparently SRP-independent, but requires a signal peptide.Ng et al. (1996) demonstrated that thehydrophobic core of the signal peptidedetermines which of the two pathways isused in yeast. The more hydrophobic thecore, the more tightly it is bound to SRP.Less hydrophobic sequences do not bindto SRP, and these preproteins use theposttranslational route.

2.2. The translocation channel

Genetic studies in yeast led to theidentification of Sec61p, a multispanningintegral membrane protein of the ERrequired for translocation of both secretoryand membrane proteins and proposed toform a channel for translocation (Deshaiesand Schekman, 1987; Stirling et al., 1992;Wilkinson et al., 1996). Sec61p interactsdirectly with Sss1p (Esnault et al., 1993;Wilkinson et al., 1997) and Sbh1p (Panzneret al ., 1995), with these three proteinsforming the Sec61p complex (Figure 2).Plath et al. (1998) proposed that Sss1pacts as a surrogate signal peptide, whichis replaced by the arrival of the signalpeptide. Yeast cells have homologues ofSec61p and Sbh1p, these being Ssh1p andSbh2p, respectively (Finke et al., 1996;Toikkanen et al., 1996), which form asecond trimer together with Sss1p in theER membrane. Because it does notassociate with an additional proteincomplex required for posttranslationaltranslocation, this complex is assumed to

Introduction

6

function solely in the cotranslationalpathway (Finke et al., 1996).

Homologues of all these proteins havebeen found in mammalian cells and theycan even function in yeast (Hartmann etal., 1994). The mammalian homologue ofSbh1p, Sec61β, facilitates cotranslationaltranslocation and interacts with the signalpeptidase (Kalies et al., 1998). Interestingly,the interaction of Sec61β with the subunitof signal peptidase complex wasdependent on the presence of membrane-bound ribosomes, suggesting that theseinteractions are enhanced whentranslocation is initiated. Sec61β has beenshown to be phosphorylated, andphosphorylation moderately stimulatedprotein translocation into the ER (Gruss etal., 1999). Phosphorylation might regulateinteractions between the channel and otherproteins involved in translocation.

In mammalian cells, a translocating chain-associated membrane protein (TRAM) isinvolved early in translocation of

polypeptides (Görlich et al., 1992a). Bysite-specific crosslinking, High et al. (1993)showed that the very N-terminal region ofthe signal peptide is in contact with TRAM,and the hydrophobic core of the signalpeptide is in contact with Sec61p. TRAMwas suggested to regulate the cytosolicexposure of secretory proteins during thetranslocational pause (Hedge et al., 1998).Yeast cells lack a homologue of TRAM.

Sec61 protein is the receptor for theribosome in the ER membrane (Figure 2).Görlich et al . (1992b) isolated frommammalian ER a membrane protein boundto ribosomes, which was a homologue ofSec61p. Similar to the yeast protein, it waslocated in the immediate vicinity of nascentpolypeptides during their membranepassage, since they could be cross-linkedto it (Mothes et al., 1994). Sec61p wasprotease-resistant in the presence ofribosomes (Kalies et al., 1994). A three-dimensional image reconstruction of aribosome bound to the yeast Sec61complex revealed an alignment of the

Figure 2. Co- and posttranslational protein translocation in yeast. In cotranslational translocationthe Sec61 complex mediates protein import into the ER. Ribosome binds tightly to the channelthereby sealing it from the cytoplasmic side. In posttranslational translocation the Sec61 complexassociates with the Sec62/63 complex to form a heptameric complex. The lumenal ATP:ase BiP/Kar2p interacts with the Sec63p and provides the energy for translocation.

7

translocation channel with the site on thelarge ribosomal unit which is believed tobe the exit site for the nascent polypeptide(Beckman et al., 1997). As the protein iselongated, it has only one way out, andtherefore, protein synthesis drivestranslocation and no other energy supplyis needed.

Electron microscopy studies verified thatthe Sec61p complex forms a channel(Hanein et al., 1996). Purified complexesfrom mammalian and yeast cells formedring-like structures in detergent with a porediameter of about 20Å. Each poreconsisted of three or four Sec61pcomplexes. Similar ring structures werealso seen in reconstituted and nativemembranes. The addition of ribosomeswas seen to increase the number of ringstructures of Sec61p complexes inreconstituted membranes (Hanein et al.,1996). The nascent chain is presumablyinserted into the channel in a loop structure(Shaw et al., 1988) (Figure 2). Song et al.(2000) noted that in the absence of afunctional Sec61 complex, the signalsequence could not be released from theSRP54 protein. They proposed that theGTP hydrolysis cycle of SRP and itsreceptor complex is regulated by the Sec61complex. The hydrophobic portion of thesignal peptide has been cross-linked tolipids (Martoglio et al., 1995), indicating thatthe channel is open laterally to lipids. Thus,hydrophobic portions of translocatingpolypeptides could reside in a hydrophobicenvironment, while the hydrophilic portionswould slide through the aqueous parts ofthe channel. Based on the measurementsof the minimal length of the translocatingpolypeptide required to bridge the distancebetween the ribosomal peptidyl transferasesite in the membrane-bound ribosomesand the lumenal active site ofoligosaccharyl transferase, Whitley et al.(1996) suggested that non-hydrophobic

nascent chains adopt a fully extendedconformation during passage through thetranslocon, whereas the hydrophobicsequence appeared to form a helicalstructure when located in the channel.

Since the ER membrane encloses a specialfolding milieu, it is important to maintainimpermeability to even small molecules.The translocation channel must open totranslocate proteins and close when thework is done. The model of cotranslationaltranslocation favoured the assumption ofa narrow channel, but Hamman et al.(1997) demonstrated with quenchingagents of different sizes that the actual sizeof the channel could expand up to 60 Å.However, because the channel isimpermeable even to ions, the opening andclosing of the channel must be tightlyregulated. Crowley et al . (1993) firstillustrated the existence of a seal byfluorescent quenching studies, in whichthey showed that small molecules from thecytoplasmic side of the membrane couldnot reach translocating polypeptidescarrying fluorecent probes. The tightjunction of the ribosome and Sec61p formsthe seal to the cytoplasmic side of thechannel. Jungnickel and Rapoport (1995)presented a model in which the signalpeptide of a nascent polypeptide isrecognized twice, first in the cytoplasm bySRP, and a second time inside themembrane by the ER translocationmachinery, Sec61p. The initial interactionbetween the ribosome-nascent chaincomplex and the Sec61p complex is weak;it is sensitive to high salt concentrations,and the nascent chain remains accessibleto added proteases. Once the nascentpolypeptide has reached a critical length,the ribosome is bound more tightly to theSec61p channel, and the polypeptide is nolonger accessible to proteases, indicatingits insertion into the Sec61p channel. Inreconstituted bacterial membranes,

Introduction

8

addition of signal peptides to thecytoplasmic site of the membrane led tothe opening of the translocation channels(Simon and Blobel, 1992). The transitionfrom weak to strong binding involves therecognition of the signal peptide, and itoccurs at the same nascent chain lengthas the opening of the channel (Jungnickeland Rapoport, 1995). The aqueoustranslocon pore is closed to the ER lumenuntil the nascent chain reaches a length ofseventy amino acids (Crowley et al., 1994).BiP, a lumenal chaperone, seals thechannel from the lumenal side of themembrane before and early in translocation(Hamman et al., 1998). The closure of thechannel requires dissociation of theribosome into its subunits (Simon andBlobel, 1991). Wang and Dobberstein(1999) characterized oligomeric complexesinvolved in translocation across the ERmembrane and could not detect any sizedifferences between the unengagedSec61p complex and the ribosome-boundone, suggesting that the Sec61p complexdoes not disassemble into its subunits aftercompletion of translocation. Takentogether, the translocation channel seemsto be a very dynamic structure.

In posttranslational translocation, theSec61p complex associates with thetetrameric Sec62/63p complex forming aheptameric complex (Deshaies et al. , 1991;Panzner et al., 1995) (Figure 2). Thecomplete heptameric complex is requiredfor posttranslational translocacation intoreconstituted proteoliposomes. Sec62pand Sec63p are essential membraneproteins, which span the membrane twoand three times, respectively (reviewed inCorsi and Schekman, 1996). The two othercomponents, Sec71p and Sec72p, are notessential for cell growth. Sec71 is a single-spanning membrane protein, and Sec72 isa peripherally associated protein on thecytosolic side of the ER membrane

(Deshaies et al., 1991; Green et al ., 1992).A Sec63p homologue has been found inmammalian cells, and it resides in the ERmembrane covered with ribosomes(Skowronek et al., 1999). The tetramericsubcomplex has been thought to replacethe functions of ribosomes and SRP.Sec62p, Sec71p and Sec72p have all beenproposed to play a role in the recognitionand binding of signal peptide (Müsch et al.,1992; Feldheim and Schekman, 1994)(Figure 2). The obvious transient nature ofthe interaction between the signal peptideand its receptors makes it dif ficult to studyin vivo . Dünnwald et al. (1999) used thesplit-ubiquitin technique to detect a possibleinteraction between Sec62p and the signalpeptide of pre-pro-α -factor. In thistechnique, the N- and C-terminal halves ofubiquitin (Ub) are fused to two test proteins,which are expected to interact. If they do,Ub is reconstituted in cells from the twohalves, and can thereafter be cleaved byUb-specific proteases, which are presentin all eukaryotic cells. This results in releaseof the reporter protein which was fused tothe C-terminal portion of Ub. The releaseof the reporter serves as a readoutindicating reconstitution of Ub. By thismethod Sec62p was shown to interact withthe signal peptide in living cells. Like theribosome, the Sec62/63p complexstimulates the assembly of Sec61pcomplex rings (Hanein et al., 1996).

Plath et al. (1998) studied the recognitionof signal peptide in posttranslationaltranslocation. By cross-linkingexperiments, they observed that the signalpeptide interacts with Sec61pindependently of ATP and Kar2p, a lumenalchaperone known to be required forposttranslational translocation. They foundthat the two transmembrane domains ofSec61p were primarily responsible for theinteraction with the hydrophobic core of thesignal peptide. The signal peptide adopts

9

a helical structure within the channel andis likely to be oriented perpendicularly tothe plane of the membrane. Cross-linksbetween the signal peptide and lipids werealso detected while the polypeptide residedin the channel, indicating that the signalpeptide binding site is located at aninterface between the channel and lipids.Matlack et al . (1997) reported thatposttranslational translocation could beobserved in detergent solution withoutmembranes. Binding of a precursor proteinwas dependent on functional signal peptideand both Sec61 and Sec62/63 complexes.

2.3. Chaperones in translocation

Molecular chaperones are defined asmolecules that prevent protein aggregationand facilitate folding by maintainingpolypeptides in productive folding pathways(Gething and Sambrook, 1992). The 70kDa class of heat shock proteins areinvolved in translocation both on thecytoplasmic and the lumenal side of the ERmembrane. They are ATPases that bindand release hydrophobic peptides (Bukauand Horwich, 1998). In yeast, cytosolicHsp70s involved in translocation are theSsa1-4 proteins (Deshaies et al., 1988),and the ER lumenal Hsp70s are Kar2p(Vogel et al., 1990), and its homologueLhs1p, which facilitates translocation of asubset of proteins into the ER (Craven etal., 1996).

Deshaies et al . (1988) studied the effect ofcytoplasmic Hsp70s on posttranslationalprotein translocation in vivo. They used ayeast strain in which all the SSA1-4 geneswere disrupted, and SSA1 was introducedto the cells in a plasmid under a galactose-regulated promotor. When the cells weregrown on glucose, the level of Ssa1pdecreased with concomitant cytoplasmic

accumulation of ER-targeted preproteins.The requirement of Ssa1p forposttranslational translocation could bebypassed if the preprotein was denatured(Chirico et al ., 1988). The conclusion fromthese studies was that the cytoplasmicHsp70s keep the polypeptide in atranslocation-competent or unfoldedconformation.

The ATPase activity of the Hsp70s isenhanced by DnaJ chaperones. Ydj1p is acytosolic yeast homologue of DnaJ (Caplanand Douglas, 1991). The temperature-sensitive mutant of Ydj1p is unable tostimulate the ATPase activity of the Ssa1p(Caplan et al., 1992), which is required forprotein release (Cyr et al., 1992). Despitethe fact that BiP and Ssa1p are 63%identical, they are unable to substitute forone another during posttranslationaltranslocation (Brodsky et al., 1993). Thiswas demonstrated to be caused by specificinteractions with unique DnaJ homologues(McCellan et al., 1998).

Since elongation and translocation ofpolypeptide chains are uncoupled inposttranslational translocation, additionalenergy is needed to drive the translocatingchain across the membrane. Themovement of the polypeptide through thetranslocation channel requires thepresence of Kar2p or its mammaliancounterpart BiP (Vogel et al ., 1990).Translocation is dependent on theinteractions of BiP/Kar2p with the lumenalJ-domain of Sec63p (Sanders et al ., 1992;Brodsky and Schekman, 1993; Scidmoreet al., 1993; Lyman and Schekman, 1995;Corsi and Schekman, 1997; Matlack et al.,1997). Each J-domain activates severalBiP/Kar2p molecules to trap neighbouringpeptides with low sequence specificity(Misselwitz et al., 1998). BiP appears tointeract with the J-domain only verytransiently, but this brief interaction is

Introduction

10

sufficient to induce the hydrolysis of ATPand to activate BiP for peptide binding. Inthe absence of peptides, BiP binds to its J-partner, indicating that BiP does not waitfor the substrate while bound to the J-domain (Misselwitz et al., 1999).

Two models have been proposed by whichBiP/Kar2p and the J-domain of Sec63pcould provide the driving force forposttranslational translocation. In one, BiP/Kar2p has an active role in stimulating theforward sliding of polypeptide by pulling thepolypeptide through the channel (Glick,1995). Alternatively, BiP/Kar2p could act asa molecular ratchet (Simon et al., 1992) bybinding to the translocating polypeptide,thereby preventing its backwardmovement. Matlack et al . (1999) showedthat bound BiP does minimize backwardmovement of the polypeptide through thechannel. In fact, antibodies against thepolypeptide could replace BiP, indicatingthat a ratchet is sufficient to achievetranslocation. However, this does notexclude the possibility of BiP functioning inthe pulling reaction as well. Additional forcemay be required if the polypeptide is in afolded conformation on the cytosolic sideof the membrane, or if cytosolic proteinsare strongly bound to it.

Although the energy needed to drivecotranslational translocation is thought tobe gained from coupling protein synthesisand translocation, mutations in BiPprevented not only posttranslationaltranslocation but also cotranslationaltranslocation (Brodsky et al., 1995). In thecase of cotranslational translocation,translocation of the last 60 amino acidscannot be driven by elongation since theyare buried in the ribosome and thetranslocase. Thus, even in cotranslationaltranslocation, completion of translocationmay be dependent on chaperones. Indeed,Nicchitta and Blobel (1993) provided

evidence that lumenal components arerequired for net transfer of secretoryproteins to mammalian microsomes.Depletion of the lumenal contents of ERmembranes by isolating them at pH 10resulted in dramatic translocation defectsin vitro . The translocating polypeptideswere inserted across the membrane andwere accessible to signal peptidase andoligosaccharide transferase, but remainedfree to pass through the membrane to thecytosolic side. The defect could becomplemented by the addition of lumenalproteins, indicating that lumenal proteinsprevent translocated polypeptides fromsliding back to the cytosol.

BiP/Kar2p has been suggested to affectevents on the cytosolic face of the ERmembrane. Mutations in BiP preventedinteraction between a polypeptide and thetranslocation channel (Sanders et al.,1992), illustratng that BiP functions earlyin translocation on the opposite face of theER membrane. BiP appeared to activatethe translocation complex. This was indeedobserved in membrane-free translocationassays; BiP was necessary for themovement of preproteins from therecognition site of Sec61p to the channelas well as for their movement through thechannel (Lyman and Schekman, 1997;Matlack et al., 1997).

2.4. Retrotranslocation

Translocation of proteins to the ER is notan irreversible process. The Sec61 channelis the channel for retrograde transport aswell. This was first observed when MHCclass I heavy chains in humancytomegalovirus-infected cells weretransported from the ER to the cytosol(Wierz et al., 1996a). This process wasshown in coimmunoprecipitation studies to

11

involve Sec61 complex, and to eventuallylead to degradation of the heavy chains bythe cytosolic proteasome (Wierz et al.,1996b). Misfolded secretory proteins in theER are also re-exported to the cytosol in aSec61p-dependent fashion (Pilon et al.,1997). Sec61p and Sss1p themselves aredegraded in the cytosol by proteasome(Biederer et al., 1996), triggered bydisassembly of the translocation complex.Like wild-type proteins, misfolded solubleproteins dissociate from the Sec61 channelafter completion of translocation (Plemperet al., 1999a) and the signal peptide isremoved (Hiller et al., 1996; Werner et al.,1996). This implies that the opening of thechannel from the lumenal side must betriggered by a mechanism that is differentfrom that used during translocation into theER.

A mutant form of carboxy peptidase Y(CPY), which is degraded by theproteasome under normal conditions, was

3. Protein maturation in the ER

found to be stabilized in the ER in sec63-1and kar2-159 cells, suggesting that theirrespective proteins have a role inretrotranslocation (Plemper et al., 1997).However, ER protein export and importseem to be mechanistically distinct, sincespecific kar2 mutations that stabilizeotherwise degradative substrates work wellin inward translocation. In addition, defectsin or depletion of Ssa1p had no effect ondegradation (Brodsky et al., 1999). As inother translocation events, in retro-translocation the channel seems to workin conjunction with other components,including ER membrane proteins Der1p,Der3p/Hrd1p and Hrd3p (Hampton et al .,1996; Knop et al., 1996; Bordallo et al .,1998). Based on genetic studies, Der3p/Hrd1p and Hrd3p functionally interact witheach other and with Sec61p (Plemper etal., 1999b).

Nascent polypeptides are modified in theER lumen. Some modifications occurduring translocation across the ERmembrane. Signal peptides are cleaved bysignal peptidase, the catalytic site of whichresides in the ER lumen (Böhni et al., 1988;YaDeau et al., 1991; Fang et al., 1997;Meyer and Hartmann, 1997).Oligosaccharides are attached to selectedresidues (Abeijon and Hirschberg, 1992;Kelleher et al., 1992), and disulfide bridgesare formed by protein disulfide isomerases(Freedman, 1984). Oligomeric proteins areassembled in the ER. All these eventsaffect protein folding, which is assisted bymolecular chaperones and foldingenzymes.

A hypothesis based on coimmuno-precipitation and cross-linking experimentspostulates that ER-resident proteinsinteract loosely with each other to form adynamic matrix (Sambrook, 1990). Thelumen of the ER contains high levels ofcalcium ions which may mediate proteininteractions. Indeed, many ER-residentproteins are calcium-binding proteins, anddepletion of calcium has been shown tocause misfolding and aggregation (Lodishet al., 1992; Suzuki et al., 1991). The matrixof ER proteins could keep newlysynthesized proteins associated to it untilfolding is completed and the affinity woulddissipate (Tatu et al., 1995).Transmembrane proteins could keep thematrix in contact with membrane sites

Introduction

12

where translocation takes place, leavingother ER areas free to accumulate foldedproteins ready for export.

3.1. Glycosylation

Oligosaccharides in the plasma membraneor cell wall and secreted glycoproteins areclassified by the nature of their linkage tothe polypeptide: the N-linked (asparagineamine) and O-linked (hydroxyl group ofserine or threonine) glycans. Thebiosynthesis of N- glycans, especially theinitial steps, is similar between species, butthe biosynthesis of O-glycans is lessconserved (Tanner and Lehle, 1987).Protein-bound oligosaccharides arebeneficial during maturation, as theyincrease solubility of folding intermediatesand stabilize the protein conformation. N-glycans also allow newly synthesizedproteins to interact with the lectin-basedchaperone system (Trombetta andHelenius, 1998).

3.1.1. N-glycosylation

N-linked glycosylation in eukaryotesinvolves two different processes: theassembly of the lipid-linked coreoligosaccharide Glc3Man9GlcNac2 at theER membrane, and the en bloc transfer ofthe oligosaccharide core from the lipiddolichol pyrophosphate to selectedasparagine residues of nascentpolypeptides. In the assembly step, at leasteleven different yeast gene products addfourteen sugars in a stepwise manner(Kukuruzinska et al., 1987). The transferstep is mediated by the oligo-saccharyltransferase complex, whichrecognizes on the nascent polypeptide asan acceptor site the consensus sequence

Asn-X-Ser/Thr, where X is any amino acidexcept proline (Kukuruzinska et al., 1987).The core oligosaccharide attached to thepolypeptide is modified in the ER lumen byremoval of three glucose and one mannoseresidues by the action of glucosidases I andII and a mannosidase (Tanner and Lehle,1987). The trimmed core oligosaccharideis then extended in the Golgi complex byaddition of mannose residues, leading tothe formation of two types of structures: theshort- and high-mannose chains. Thedegree of protein glycosylation is controlledin cells, since not all potential glycosylationsites are utilized. Glycosylation may becompeted by protein synthesis,translocation and folding kinetics. Inmammalian cells, a ribosome-associatedmembrane protein, RAMP4, was reportedto regulate N-glycosylation bytranslocational pausing (Schröder et al .,1999).

3.1.2. O-glycosylation

In yeast, O-glycosylation is initiated in theER (Haselbeck and Tanner, 1983). Dolicholmonophosphate is the donor of the firstmannose residue transferred to the hydroxygroups of serine and threonine (Tanner andLehle, 1987; Immervoll et al., 1995; Lussieret al ., 1995). Although the consensussequence for O-glycosylation in yeast hasyet to be determined, a proline residue inthe vicinity of serine or threonine mayenhance the first mannosylation reaction.The elongation of the O-linked chain occursin the Golgi. O-linked chains areunbranched and contain at most four to fivemannosyl residues (Lehle and Bause,1984).

13

3.2. Lectin-like chaperones: Calnexinand calreticulin

Calnexin couples N-glycosylation of newlysynthesized proteins with their productivefolding in the ER (Bergeron et al., 1994).Calnexin is an integral type I ER membraneprotein, which was originally identified as amajor calcium-binding protein of themammalian ER (Wada et al., 1991). Thesubstrate-binding domain resides in thelumen. The ER-localization signal RKPRREresides at the C-terminus. Its cytoplasmictail contains a phosphorylation site(Bergeron et al ., 1994), andphosphorylation was shown to lead toincreased association of calnexin withmembrane-bound ribosomes (Chevet etal ., 1999). Coimmunoprecipitationexperiments in the presence of the N-glycosylation inhibitor tunicamycindemonstrated that calnexin binds to N-glycosylated proteins (Ou et al., 1993). Thebinding preference for glycoproteins isbased on a lectin-like affinity formonoglucosylated N-linked oligo-saccharides (Hammond et al., 1994).Moreover, calnexin has been shown in vitroto act solely as a lectin, since binding ofmonoglucosylated Rnase B –protein wasindependent of the conformation of theglycoprotein (Zapun et al ., 1997).Calreticulin, a soluble luminal homologueof calnexin, was shown to bind similarly tounfolded glycoproteins (Spiro et al., 1996).A calnexin homologue, the product ofCNE1 gene in yeast, is not essential forviability (Parlati et al ., 1995). It may,however, function in quality control similarto its mammalian counterparts. Forexample, in ∆cne1 cells, a temperature-sensitive mutant protein that is retained inthe ER in wild type cells, was secreted(Parlati et al., 1995).

The monoglucosylated N-linkedoligosaccharides arise as transientintermediates during the processing of N-glycans in the ER (Ware et al., 1995; Spiroet al ., 1996). These structures aregenerated either by the action ofglucosidases I and II or by glucosyl-transferase re-adding a glucose residue tothe trimmed glycans (Helenius et al., 1997).Helenius et al. (1997) proposed a qualitycontrol function for calnexin and calreticulin.U D P - g l u c o s e : g l y c o p r o t e i nglucosyltransferase (UGGT) wassuggested to be a folding sensor: if theconformation of the glycoprotein was notnative, it would re-add the glucose residue.This enables the incompletely foldedprotein to rebind to calnexin or calreticulin.The on and of f cycle would proceed untilfolding is complete. More recent in vivostudies with thermosensitive viralglycoprotein have supported this model(Cannon and Helenius, 1999).

The mannose residues of the N-linkedsugars have been proposed to set a timelimit for folding. Misfolded glycoproteinsare retained in the ER by the UGGT-calnexin-glucosidase II cycle, which leadsto the complete trimming by mannosidases(Helenius et al., 1997). Jakob et al. (1998)demonstrated that the number and linkageof mannose residues of an N-linkedoligosaccharide determine the degradationof misfolded proteins. Deletion ofmannosidases Mns1p, Alg9p and Alg12pled to reduced degradation of a mutatedcarboxy peptidase Y (CPY). It wasspeculated that a lectin-like receptor wouldrecognize the trimmed oligosaccharide andtogether with a chaperone such as BiPwould guide the misfolded protein toretrotranslocation and subsequentdegradation in the cytosol by theproteasome machinery. Yeast KRE5 is ahomologue of the mammalian UGGT(Meaden et al., 1990). However, no

Introduction

14

glucosyltransferase activity for this yeastprotein has been demonstrated fo far(Jakob et al ., 1998). However,monoglucosylated N-linked sugar chains ofglycoproteins were found to reduce thelevel of unfolded protein in the ER undermild reducing conditions (Jakob et al .,1998).

In addition to its role in proofreading ofglycoproteins, calnexin may have anotherfunction in mammalian cells. It has beenreported to bind unglycosylatedpolypeptides (Arunachalam and Cresswell,1995; Kim and Arvan, 1995). This was,however, suggested to be caused byaggregation (Cannon et al., 1996).Recently, calnexin was identified as apeptide-binding protein and postulated tohave a role in the removal and degradationof peptides in the ER (Spee et al., 1999).

3.3. Classical chaperones

The nascent polypeptide encounters anumber of molecular chaperones andfolding enzymes upon entering the lumenof the ER and begins to fold, in some cases,even before translation is completed(Hammond et al., 1994). Among the mostabundant and best-characterizedchaperones in the ER is BiP. As an Hsp70protein, it consists of two domains, a highlyconserved N-terminal ATP-binding domainand a C-terminal peptide- binding domain.BiP associates transiently with numerousproteins during folding and binds morepermanently to misfolded proteins andincompletely assembled oligomers(Gething and Sambrook, 1992). It wasoriginally identified as a protein bindingnoncovalently to free immunoglobulinheavy chains (Haas and Wabl, 1983).

BiP binds to unfolded nascent polypeptides(Simons et al ., 1995; Hendershot et al .,1996). In vitro binding studies with a set ofpeptides of random sequence but a definedchain length revealed that BiP recognizesheptapeptides of aliphatic residues (Flynnet al., 1991). The same kind of peptidecharacteristics for BiP binding were foundby af finity screening of a peptide libraryexpressed on the surface ofbacteriophages (Blond-Elguindi et al.,1993). Furthermore, the aliphatic residueswere preferred in an alternating manner,suggesting that the extended sequence, inwhich hydrophobic residues face the samedirection, would fit into the peptide-bindingpocket of BiP (Blond-Elguindi et al., 1993).The crystal structure of the peptide-bindingdomain of the bacterial Hsp70 homologueDnaK with a bound peptide has beendetermined. The peptide resides in thebinding pocket in an extendedconformation (Zhu et al., 1996). It has beenspeculated that aliphatic peptide residueswould occur approximately every sixteenthresidue in an average globular protein(Flynn et al., 1991), enabling the binding ofBiP to the vast majority of secretoryproteins. In spite of this, some secretoryproteins do not associate with BiP in normalconditions (Morris et al., 1997).

Like all Hsp70 proteins, BiP has a highaffinity for ATP but a low turnover number(Kassenbrock and Kelly, 1989). The bindingand hydrolysis of ATP is coupled to peptidebinding and release. ADP binding stabilizespeptide binding, whereas ATP bindinginduces conformational changes in thepeptide-binding domain, causing therelease of bound peptides (Palleros et al.,1993).

Yeast KAR2, encoding a homologue of BiP,was isolated as a gene required for nuclearfusion after conjugation of haploid cells and

15

shown to be essential for viability(Normington et al., 1989; Rose et al., 1989).Expression of both mammalian and yeastBiP is induced by accumulation of unfoldedproteins in the ER (Gething and Sambrook,1992; Rose et al., 1989). In addition, theexpression of yeast KAR2 is regulated byheat shock (Rose et al., 1989; Mori et al.,1992; Kohno et al., 1993).

Yeast ER contains another Hsp70-relatedprotein, Lhs1p (also called Cer1p or Ssi1p)(Baxter et al., 1996; Craven et al., 1996;Hamilton and Flynn, 1996). In addition toits role in facilitating translocation, it hasbeen suggested to have chaperoningactivity, particularly at lower temperatures(Hamilton et al., 1999). Saris et al. (1997;1998) have shown that Lhs1p is requiredin yeast ER for refolding, stabilization andacquisition of secretion competence ofheat-denatured proteins. Unlike BiP, Lhs1pis not essential under normal conditions,but is required for acquisition ofthermotolerance (Saris et al., 1997). Itappears to belong to a heat-resistantsurvival machinery enabling yeast cells torecover from severe heat stress (Jämsä etal., 1995b; Saris et al., 1997; 1998).Mammalian cells have another abundantER protein, GRP94, which interactstransiently with unfolded proteins (Melnicket al., 1994). The function of this protein isthought to be similar to that of BiP.

3.4. Folding enzymes: Proteindisulfide isomerases and peptidylprolyl isomerases

Disulfide bonds formed in the reducingenvironment of ER lumen stabilize thethree- dimensional structure of numeroussecretory proteins and the quaternarystructure of some protein complexes.

Enzymes that catalyze the formation and/or rearrangement of these bonds are calledfolding enzymes.

Protein disulfide isomerase (PDI) belongsto the thioredixin superfamily and catalyzesboth the isomerization and formation ofdisulfide bonds (Freedman,1984). As ahighly abundant ER luminal protein, itconstitutes nearly 1% of the total cellularproteins. It is highly conserved betweenspecies. Quite surprisingly, PDI has fourthioredoxin-like folds, even though only twoof the domains display sequence homologyto thioredoxin (Kemmink et al.,1997). Theprotein has two catalytic sites, doublecysteines, in two thioredoxin-relateddomains, which are separated by twosimilar domains lacking redox-active sites(Edman et al., 1985; Freedman et al., 1994;Kemmink et al .,1997). PDI has beenisolated as a homodimer (Freedman et al.,1994). In yeast, PDI is essential for cellsurvival (Scherens et al., 1991), and itsforemost function is to reorganize disulfidebonds of non-native proteins (Laboissiereet al., 1995).

The isomerase activity of PDI, where PDIis transiently bound via mixed disulfide toits substrate, resembles the function ofchaperones in keeping the polypeptide ina folding- competent stage. The fullisomerase activity of PDI requires thepresence of domains lacking the catalyticsite (Darby et al., 1998). Interestingly, oneof these domains provides the core ofpeptide-binding ability to PDI (Klappa et al.,1998). An unfolded protein responseelement (UPRE) was found in the promoterof PDI1, supporting PDI’s function as achaperone (Shamu et al., 1994). Thefunction of PDI has recently been linked tothe quality control machinery; it has beenshown to be required for export anddegradation of a cysteine-free misfolded

Introduction

16

secretory protein from the endoplasmicreticulum (Gillece et al., 1999).

Oxidized gluthathione was proposed to bethe source of the oxidizing equivalentsneeded to generate disulfide bonds in theER (Hwang et al., 1992). However, mutantyeast cells lacking gluthathione were shownto be capable of efficient disulfide bondformation (Frand and Kaiser, 1998). Ero1p,a novel essential protein in the ERmembrane, was demonstrated to beresponsible for maintaining PDI in anoxidized state (Pollard et al., 1998; Frandand Kaiser, 1999).

Cooperation between different ER proteinscreates additional functions. ERp57, amember of the protein disulfide isomerasefamily, has been cross-linked to partiallydeglucosylated glycoproteins (Oliver et al.,1997). Calnexin and calreticulin presentglycoproteins lacking disulfides to ERp57,which catalyzes the formation of thedisulfide bond, thus enhancing folding(Zapun et al., 1998). Interaction of both thesubstrate and the enzyme with the lectinbrings them into close proximity, thusenhancing the catalytic activity of Erp57.Other PDI-like proteins may functionsimilarly with other ER proteins.

During protein synthesis at the ribosomesmost peptide bonds are connected in thetrans conformation. The trans conformationis often remained in native proteinstructures. The peptidyl-prolyl bond,however, can exist in cis conformation aswell. The isomerization reaction was rate-limiting in refolding experiments (Kiefhaberet al ., 1990). Since the foldingintermediates are sensitive to proteolyticdigestion and aggregation, an enzymecatalyzing the reaction was proposed toexist. Peptidyl prolyl isomerases (PPIs)catalyze the isomerization of cis and transpeptide bonds on the N-terminal side ofproline residues. These enzymes are foundin all cellular compartments where proteinfolding occurs. PPIs are members of threefamilies, the cyclophilins, the FK-bindingproteins (FKBPs) and the parvulins (Ruddet al., 1995). Accumulation of unfoldedprotein in the yeast ER induces expressionof FKBP FPR1 (Partaledis and Berlin,1993), suggesting their role in proteinfolding in vivo .

17

AIMS OF THE STUDY

Translocation of secretory proteins into theendoplasmic reticulum initiates theirjourney through the secretorycompartments to the extracellular milieu.In yeast, proteins are translocated into theER either simultaneously with proteinsynthesis, cotranslationally, or after proteinsynthesis is completed, posttranslationally.More than two decades of research oncotranslational translocation has led to adetailed picture of this process, whilepieces of the mechanism ofposttranslational translocation are stillmissing. Translocating polypeptidesencounter a number of molecularchaperones and folding enzymes on thelumenal side of the ER membrane. Theaims of this study were to expand on themechanism of posttranslationaltranslocation in living Saccharomycescerevisiae cells, with particular emphasison the cytoplasmic events before the actualcrossing of the membrane, and to studythe role of molecular chaperones intranslocation as well as in folding.

The specific aims were:

I: To study the conformational stage ofsecretory proteins on the cytosolic side ofthe ER membrane before theirposttranslational translocation into the ER,

II: To determine, whether a folded secretoryprotein could be accommodated by thetranslocation channel and inserted into theER, and

III: To study the role of ER-locatedchaperone BiP in folding of nascentpolypeptides and to determine, whether itsfunctions in translocation and folding areseparate.

Aims of the study

18

MATERIALS AND METHODS

The yeast strains used in this study arelisted in Table 1, and the defects of mutantsare described in Table 2. The usedexperimental methods are listed in Table

3. The detailed description of each methodis found in the original publications. Thereporter proteins are schematicallypresented in Figure 3.

Table 1. The yeast strains used in this study.

19

Table 3. The methods used in this study.

Table 2. Relevant defects of the mutant yeast strains.

Materials and methods

20

Figure 3. Schematic presentation of the reporter proteins used in this study. A) Hsp150∆-β-lactamase, B) ∆1-18Hsp150∆-β-lactamase and C) Hsp150∆-NGFR

e. The Hsp150∆ consists of

subunit I (horizontally striped) and an N-terminal part of the subunit II composed of a repetetiveregion (11 white boxes) and part of a unique C-terminus (diagonally striped box). The signalpeptides are indicated by black boxes, the cysteine residues by stars and the potential N-glycosylation site of NGFR

e by N. An arrow indicates the site of mutation, in which glutamic acid

166 is replaced either with alanine or aspartic acid. Hsp150∆ contains 95 potential O-glycosylationsites.

21

Hsp150 is a yeast secretory glycoprotein(Russo et al., 1992) consisting of acleavable signal peptide of 18 amino acids,subunit I and subunit II. The two subunitsare separated by a Kex2 recognition site,which is presumably cleaved in the Golgi.Subunit II contains a region of 19 aminoacids, which is repeated 11 times, and aunique C-terminal region containing fourcysteines, which form at least oneintrachain disulfide bond (Jämsä et al.,1994). The promoter of the HSP150includes heat-inducible elements (Russo etal., 1992). An N-terminal fragment ofHsp150, Hsp150D, was used as a carrierin all fusion proteins in this study. It consistsof the signal peptide for ER targeting,subunit I and the repetitive region of subunitII (Jämsä et al., 1995a). The carrierfragment has 95 potential O-glycosylationsites, but not a single N-glycosylation site(Russo et al., 1992). All the O-glycosylationsites of the first 53 amino acids of Hsp150are utilized (Suntio et al. 1999). When fusedto E. coli β-lactamase or the extracellulardomain of rat nerve growth factor receptor(p75) (NGFR

e), it has been shown to confer

secretion competence to both heterologousproteins (Simonen et al., 1994; 1996).

To study posttranslational proteintranslocation into the ER and folding inliving yeast cells, four different Hsp150∆fusion proteins were used. E. coli β-

lactamase was chosen as a reporter,because it has enzymatic activity thatreflects its conformation and is easy todetermine. Furthermore, in its authenticform, it is trypsin-resistant and has aglobular structure with a single disulfidebond near the active site (Jelsch et al.,1992). Two point-mutated β-lactamaseshaving decreased deacylation reactionvelocities with antibiotics (Adachi et al.,1991) also were fused to the Hsp150Dcarrier (II). The crystal structure of themutant β-lactamase which irreversiblybinds benzylpenicillin has been solved(Strynadka et al ., 1992). Based on highsequence homology with the tumournecrosis factor receptor ectodomain(TNFR

e) (Radeke et al., 1987) and the

crystal structure of TNFRe (Banner et al .,

1993), NGFRe is likely to have an extended

rod-like structure of four domains, each ofwhich have three intradomain disulfidebonds (III, Figure 2). NGFR

e has one N-

glycosylation site in the first domain, whichis glycosylated in roughly half of themolecules in the yeast ER (Simonen et al.,1996). The high number of cysteines anddisulfide bonds of NGFR

e were anticipated

to challenge the folding apparatus in yeastER.

1. The reporter proteins: Hsp150 and the fusion proteins

RESULTS

Results

22

2.1. Hsp150∆∆∆∆∆ is translocatedposttranslationally (I)

According to the hydrophobicity plot of thesignal peptide (Ng et al., 1996), Hsp150was thought to use the posttranslationaltranslocation pathway. This was verified bytaking advantage of specific sec mutantshaving defects in either co- orposttranslational translocation. sec62-101cells are constitutively defective inposttranslational translocation (Ng et al.,1996). The strain expressing Hsp150∆-β-lactamase was pulse-labelled with[35S]methionine/cysteine and chased withcycloheximide (CHX). After immuno-precipitation and SDS-PAGE, no proteincould be seen in the culture mediumsamples even after 120 minutes of chasing(I, Figure 1). Instead, a 66 kDa protein wasdetected in cell lysates. The O-glycosylatedER form of Hsp150∆-β-lactamase hasbeen shown to migrate as a 110 kDa bandin SDS-PAGE (Simonen et al., 1994). Thesmaller 66 kDa band was therefore likelyto present the unglycosylated precursor ofthe fusion protein. In similarly labelled andchased wild-type and sec65-1 strains,Hsp150∆-β-lactamase was secreted intothe culture medium. In the sec65-1 mutant,the cotranslational pathway is blocked at atemperature of 37°C due to thenonfunctional SRP (Stirling et al., 1992).Thus, the signal peptide of Hsp150 usesthe posttranslational pathway to enter theER lumen.

2.2. The translocation intermediate onthe cytoplasmic face of the ERmembrane (I)

To study the topology of the 66 kDa protein,experimental conditions were sought toretard the translocation process but not tototally inhibit it. As posttranslationaltranslocation requires ATP (Vogel et al .,1990), a decrease of the ATP level waspresumed to slow down translocation.Indeed, in labelling experiments at high celldensities, where the availability of glucosebecame limiting, more 66 kDa form couldbe detected than with low cell densitysamples (I, Figure 2). To confirm that the66 kDa form was capable of translocatingacross the membrane, sec18-1 cells inwhich the fusion of secretory vesicles tothe Golgi is prevented at the restrictivetemperature were labelled and chasedunder high cell density conditions. Duringthe chase, the 66 kDa form was convertedto the glycosylated form of 110 kDa in 20minutes (I, Figure 3A). The translocationof authentic Hsp150 was not retarded athigh cell densities (I, Figure 3B), as theunglycosylated form of 50 kDa could hardlybe detected even after a one-minute pulse(data not shown). Thus, the unglycosylatedform of the fusion protein could be detectednot only in cells having a translocationaldefect, but also as an intermediate form incells which were wild-type for translocation.

To reveal the topology of the different formsof Hsp150∆-β-lactamase, microsomeswere isolated after incubation of sec18-1cells at the restrictive temperature underhigh cell density, and subjected to SDS-PAGE and Western blot analysis with anti-β-lactamase antiserum. Both the 66 kDaform and the ER form of 110 kDa could bedetected (I, Figure 4). The 66 kDa form was

2. Protein folding before translocation into the ER ( I )

23

sensitive to trypsin digestion, with theconcomitant increase of a 32 kDa proteinfragment. When digestion with trypsin wasdone in the presence of Triton X-100 tosolubilize the membranes, the 110 kDaform also disappeared and theimmunoreactive 32 kDa band wasdetected. As the digestion of the 66 kDaform occurred in the absence of Triton X-100, it was likely to reside on thecytoplasmic side of the ER membrane. Theappearance of the 32 kDa fragmentsuggested that the β-lactamase portion ofthe fusion protein on both sides of the ERmembrane was probably folded, since theauthentic protein has the same size and isknown to be trypsin-resistant (Minsky et al.,1986). In contrast, the Hsp150 portion ofthe fusion protein was presumed to betrypsin-sensitive due to its many recognitionsites and lack of a regular secondarystructure (Jämsä et al., 1995a). Similarresults were obtained when the digestionexperiments were performed withmetabolically labelled whole cell lysates (I,Figure 5).

2.3. The translocation intermediate isactive (I)

To gain further information about theconformation of the β-lactamase portion ofthe cytoplasmic fusion protein, the activity

of β-lactamase was measured in sec63-1cells, where posttranslational translocationis blocked at the restrictive temperature(Rothblatt et al., 1989). At 37°C, activityaccumulated inside the cells, whereasnothing could be detected in the medium(I, Figure 7 B). After addition of CHX, thecells were shifted to the permissivetemperature of 24°C. The activity inside thecells decreased with time and wasconcomitantly secreted to the medium. Thereversal of the sec63-1 block was notcomplete, as revealed by a pulse-labellingexperiment (I, Figure 6). This explains thelower activity in the medium samples ofsec63-1 cells as compared with sec18-1cells after chasing at the permissivetemperature. In contrast to the activity ofβ-lactamase in the ER lumen (I, Figure 7A),the cytoplasmic activity was unaffected bythe reducing agent DTT (I, Figure 7B),which diffuses across the membranes andprevents the formation of disulfide bridgesand reduces the existing ones (Jämsä etal., 1994). Thus, the reporter protein foldedto a active conformation in the cytoplasmprior its translocation into the ER, probablyeven without the formation of the disulfidebond. This conclusion was supported bythe similarity of K

m values for nitrocefin of

cytoplasmic and ER forms of Hsp150∆-β-lactamase to those of authentic E. coli β-lactamase (I, Table 2).

3. The conformation of proteins attached to the translocon (II)

3.1. The β-lactamase mutants (II)

Above I showed that the β-lactamaseportion of newly synthesized Hsp150∆-β-lactamase folded first into a native-likeconformation in the yeast cytoplasm andwas thereafter translocated. The question

thus arises, whether it passed thetranslocon in a folded form or whether itfirst had to be unfolded. β-lactamaseshydrolyze β-lactam rings of antibiotics. Thereaction occurs through an acyl-enzymeintermediate, in which the enzyme and thesubstrate are covalently bound to each

Results

24

other. To find out whether the foldedHsp150∆-β-lactamase had to unfold beforetranslocation, we took advantage of two β-lactamase mutants which bind to antibioticsin an irreversible and reversible manner.In these mutants, glutamic acid Glu166,which resides at the bottom of the activesite of β-lactamase, was mutated to alanine(E166A) or aspartic acid (E166D). Acovalent, stable complex with the β-lactamantibiotic, benzylpenicillin (PenG),accumulates in E166A mutants, originallycreated for studying the catalyticmechanism of the hydrolysis reaction.However, PenG bound to the E166Dmutant decays slowly and the hydrolyticactivity is retained (Adachi et al., 1991).

3.2. The effect of PenG on the reporter(II)

The two mutant forms of β-lactamase werefused to the Hsp150∆-carrier to create thefusion proteins E166A and E166D, andexpressed in yeast strains, which are wildtype for secretion. The ERG6 gene, whoseproduct functions in sterol synthesis, wasdisrupted to change the sterol compositionof membranes to allow the penetration ofdrugs (Jackson and Képès, 1994). Thecells were preincubated with PenG andpulse-labelled with [ 35S] -methionine/cysteine. The majority of the fusion proteinE166A was found in cell lysates in acytoplasmic 66 kDa form (II, Figure 1A),and very little was in the medium. Thecytosolic form did not disappear during thechase with CHX. In the absence of PenG,the E166A fusion protein was translocatedand secreted normally, indicating that themutation alone did not affect translocationof the fusion protein (II, Figure 1B).However, the mutation inactivated thefusion protein (II, Figure 2). Thus, PenG,by binding irreversibly to the mutated β-

lactamase portion of the fusion protein,presumably prevented its unfolding andthereby its translocation into the ER.

3.3. Subcellular localization of PenG-bound reporter protein (II)

Next, we wanted to verify that the E166Afusion protein was on the correct exportpathway. The cells harbouring the mutatedfusion protein were incubated with PenGand then subjected to immunofluorescentstaining either with β-lactamase antiserumor anti-Lhs1 antibody to mark the ER.These two antibodies revealed very similarstructures (II, Figure 3 A). In yeast, the ERmostly resides beneath the plasmamembrane. E166A bound to the PenG wasthus likely to reside on the ER membrane.The Hsp150∆-β-lactamase lacking a signalpeptide resided in the cytosol (II, Figure3Ac), indicating that the association ofE166A with the ER membrane wasdependent on the signal peptide.

Hence, it follows that translocationchannels should be saturated in vivo duringthe expression of the mutated fusionprotein in the presence of PenG. To seewhether this was the case, the translocationof ER-resident protein BiP was studied.Unlabelled E166A fusion protein wasexpressed in the presence of PenG atdifferent times. Then, the cells weremetabolically labelled and chased. The celllysates were subjected to immuno-precipitation with anti-BiP antiserum andSDS-PAGE. The longer the accumulationof the unlabelled PenG-bound fusionprotein, the more pre-BiP was detected andthe less mature form was generated (II,Figure 4). The synthesis of pre-BiP wasfound to decrease concomitantly with theaccumulation time of E166A fusion proteinbound to PenG. Thus, the accumulation of

25

the unprocessed precursor form of themutated fusion protein in the presence ofPenG led to the saturation of translocationchannels, demonstrating that it was boundto functional translocons. As BiP uses thecotranslational pathway to enter the ERlumen (Ng et al., 1996), the same Sec61pcomplexes seem to be used by substratesof both translocation pathways in vivo.

The extent of translocation of the mutatedfusion protein was then studied. E166A waslabelled in the presence of PenG andsubjected to immunoprecipitation and SDS-PAGE. Native Hsp150∆-β-lactamase wassimilarly treated in sec63-1 cells, at therestrictive temperature to block itstranslocation. These two proteins migratedsimilarly and slightly more slowly than theHsp150∆-β-lactamase variant lacking thesignal peptide (II, Figure 3 B). Furthermore,E166A could not be labelled with mannosein the presence of PenG ( II, Figure 3B).Taken together, the E166A fusion proteinbound to PenG was associated withfunctional translocons on the ERmembrane, but was not inserted far enoughto be reached by the signal peptidase orO-glycosylation apparatus.

3.4. The effect of reversibly bounddrug on translocation (II)

The experiments with the Hsp150∆-β-lactamaseE166D mutant, which bindsPenG in a reversible manner (Adachi et al.,1991), supported the view that theirreversible folding of the β-lactamaseportion of the fusion protein resulted in itsaccumulation on the cytosolic face of theER membrane. The translocation efficiencyof the E166D mutant was found not to beaffected by PenG (II, Figure 1 C and D).Neither was the translocation of nativeHsp150∆-β-lactamase affected by another

antibiotic, Cloxacillin (Clx), which ishydrolyzed by the enzyme (Citri and Zyk,1982). That reversible binding allowedtranslocation indicates that the ligand hadto be displaced for translocation.

3.5. An unfolding step prior totranslocation (I, II)

Since the release of the ligand allowedtranslocation across the ER membrane, wesuggest that unfolding ofpretranslocationally folded domains isrequired for successful translocation. Todetermine the putative unfolding step,Hsp150∆-β-lactamase was expressed inthree dif ferent strains deficient intranslocation, namely sec61-41, sec63-1and kar2-159. In the first, docking ofsecretory protein to the translocon occurs,but the precursor is not inserted on thechannel at the restrictive temperature of17°C (Pilon et al., 1998). The latter two aredefective in posttranslational translocation.In kar2-159 cells, BiP is not functional atthe restrictive temperature (Vogel et al .,1990), and in sec63-1, the J-domain of theprotein is unable to stimulate the ATPaseactivity of BiP (Lyman and Schekman,1997). At 37°C, translocation was ratherslow in sec61-41 cells (II, Figure 5), whichallowed the use of this same temperaturefor all of these strains. As revealed byimmunoprecipitation and SDS-PAGE, in allstrains the amounts of fusion proteinaccumulated on the cytosolic face of theER membrane were similar (II, Figure 5A).This was also observed in Western blottinganalysis of isolated microsomes. Thus, wecould compare the activities of β-lactamasein these strains to gain information on theirconformations.

Results

26

The cytoplasmic activities of β-lactamaseincreased in sec63-1 and kar2-159 cells ina similar manner during incubation at 37°C.In contrast, the activity in sec61-41 cellsremained at a low level throughout theexperiment (II, Figure 5B). Since there wasno difference in protein expression in thesestrains, most of the fusion protein in thesec61-41 cells, in which translocationchannels were incapable ofaccommodating polypeptides, wasapparently in a non-native conformation.The inactivity does not, however, tell usmuch about the conformational stage of theprotein, as even a small change in its three-dimensional structure could lead toinactivation. To obtain further information,the T

m values and protease-resistance of

Hsp150∆-β-lactamases in the sametranslocation-defective cells weredetermined.The T

m value for protein in

sec61-41 cells was lower than that insec63-1 cells (Figure 5C) and for previouslymeasured native Hsp150∆-β-lactamase(Holkeri and Makarow, 1998). For theprotease-resistance assay, these cellswere metabolically labelled at the restrictivetemperature, Hsp150∆-β-lactamase wasimmunoprecipitated in non-denaturingconditions and then subjected to trypsindigestion. In sec63-1 cells, the β-lactamaseportion of the fusion protein remained

stable in the presence of trypsin (II, Figure6A). In contrast, in sec61-41 cells, most ofthe fusion protein was degraded under thesame conditions( II, Figure 6A). Thus, insec61-41 cells, where the fusion proteininteracted with the translocation channelbut was not allowed to penetrate throughthe pore, it seemed to be unfolded to atrypsin-sensitive form. The translocationdefect in sec61-41 cells was reversible,since during the chase in the presence ofCHX at the permissive temperature, thefusion protein was secreted to the culturemedium (II, Figure 7).

To study the possible unfolding machineryin the yeast cytoplasm, the 66 kDacytoplasmic form and the 110 kDa ER formof the reporter protein were accumulatedin sec62-101, sec63-201 and sec18-1cells, and subjected to coimmuno-precipitation with anti-Hsp70 antibody. TheER form accumulating in sec18-1 cells atthe restrictive temperature was used as areference. The cytoplasmic reporter proteinwas found in association with cytosolicHsp70 chaperones (I, Figure 8), whereasthe 110 kDa form of the reporter was not,indicating the specificity of the interaction.Preimmunoserum precipitated neither thereporter protein nor Hsp70.

4. Protein folding in the ER (III)

4.1. The effect of DTT on secretion (III)

In the preceding section, the folding ofpreproteins in the cytoplasm wasdescribed. Now we move to the other sideof the ER membrane to study proteinfolding. The reducing agent, DTT, has beenshown to penetrate yeast cell walls andmembranes and to inhibit disulfideformation in the ER in a reversible manner(Jämsä et al., 1994). Many reduced

secretory proteins and the Hsp150∆-β-lactamase fusion protein are known toremain in the ER in the presence of DTT(Jämsä et al., 1994). The requirement ofBiP for translocation of proteins into the ERhas made it dif ficult to get in vivoinformation about its role in protein folding.The DTT-dependent secretion block wastaken advantage of in studying therequirement of BiP for conformationalmaturation of two reporter proteins,

27

Hsp150∆-β-lactamase and Hsp150∆-NGFR

e.

First, the effect of DTT on secretion ofHsp150∆-NGFR

e was established. In non-

reducing SDS-PAGE, native and reducedmolecules migrate dif ferently due toconformational differences. Hsp150∆-NGFR

e was expressed and metabolically

labelled with [ 35S]-methionine/cysteine insec18-1 cells at the restrictive temperatureof 37°C, where secretory proteinsaccumulate in the ER and ER-derivedvesicles (Kaiser and Schekman, 1990), inthe presence or absence of DTT (III, Figure3B). When the migration rates in non-reducing SDS-PAGE were compared afterimmunoprecipitation with anti-Hsp150∆-NGFR

e antiserum, the DTT-reduced fusion

protein appeared to migrate more slowlythan the native fusion protein. The removalof DTT by washing and chasing with CHXin the absence of DTT at the restrictivetemperature resulted in molecules havingsimilar migration rates to native fusionproteins. Thus, disulfide bond formation,occurring normally at 37°C, was preventedin the presence of DTT, and the reducedmolecules were reoxidized in the ER afterremoval of DTT.

To study whether the reoxidized Hsp150∆-NGFR

e molecules could be secreted, a

similar labelling experiment was done inwild type cells (III, Figure 3A). Without DTT,the fusion protein was secreted to theculture medium. The addition of DTT nearlytotally inhibited the secretion of the fusionprotein, as very little protein could bedetected in the culture medium. When DTTwas removed, the fusion protein was againsecreted. DTT treatment reversibly blockedthe secretion of the fusion protein. Afterremoval of the DTT-imposed secretionblock, the secreted Hsp150D-NGFR

emolecules migrated more slowly than the

native molecules in SDS-PAGE. Thereduced molecules apparently were moreeffectively N-glycosylated.

However, in wild-type cells the DTT-treatedmolecules were only weakly detectable,raising the possibility that they could havebeen heterogenously N-glycosylated in theGolgi. To confirm that DTT-treatmentprevented secretion from the ER, labellingwas performed in wild-type cells in thepresence of both DTT and tunicamycin(TM) to inhibit N-glycosylation (III, Figure4). The fusion protein was now clearlyvisible and migrated similarly as the ER-retained form of the fusion protein in sec18-1 cells. The Golgi form of the fusion proteinin sec7 cells at the restrictive temparaturemigrated more slowly and similarly to themature, secreted form of the fusion protein.Since the fusion protein in wild type cells inthe presence of DTT apparently lackedextended glycans, it was likely to reside inthe pre-Golgi compartment. Its poordetection can be due to the reduced affinityof antibodies to DTT-treated molecules,which have an altered conformation.

Next, the kinetics of resumption of asecretion-competent conformation wasstudied for the two reporter proteins. Wildtype cells expressing Hsp150∆-NGFR

ewere pulse-labelled with DTT and chasedfor different times after removal of DTT.Hsp150∆-NGFR

e was secreted to the

culture medium very rapidly, in less thanfive minutes (III, Figure 7A). The additionof TM had no effect on the rate of secretion.Hsp150∆-β-lactamase, similarly expressedand labelled, was secreted in about 40minutes after re-establishing of oxidativeconditions in the ER (III, Figure 7C). It thusrequired a considerably longer time forfolding into a secretion-competentconformation after reoxidation than theNGFR

e fusion protein.

Results

28

4.2. The role of BiP in conformationalmaturation and translocation (III)

To study the chaperoning role of BiP, theHsp150∆-NGFRe reporter protein wasexpressed in temperature-sensitive kar2-159 cells, in which BiP is irreversiblyinactivated at the restrictive temperature of34°C (Vogel et al., 1990). The translocationblock was circumvented by using DTT atpermissive temperature to prevent disulfideformation and secretion during labellingwith 35S-methionine/cysteine, but nottranslocation into the ER, and thereafterdiluting DTT at the nonpermissivetemperature to inactivate the function ofBiP, followed by chase in the presence ofCHX for different times. Reference cellswere treated similarly at the permissivetemperature. At both temperatures, thereporter protein could be found in theculture medium (III, Figure 5A). Theaddition of TM had no effect on secretionof the reporter protein (III, Figure 5B). Inboth cases, the kinetics of secretionseemed to be similar. Hsp150∆-NGFRe

thus acquired disulfides and folded into asecretion- competent conformation in theabsence of functional BiP and N-glycosylation.

The same experiment was performed onHsp150∆-β-lactamase. Without functionalBiP, secretion of reduced and reoxidized

Hsp150∆-β-lactamase was severelyimpaired (III, Figure 6A). Theconformational maturation of a yeastvacuolar protein CPY has been previouslyreported to be similarly dependent on thefunction of BiP (Simons et al., 1995). In thevery same kar2-159 cells, the maturationof CPY indeed required BiP, whereas thematuration of the Hsp150∆-NGFR

e did not

(III, Figure 6B). Thus, both CPY andHsp150∆-β-lactamase needed help fromBiP for conformational maturation afterremoval of DTT. Under the sameconditions, Hsp150∆-NGFR

e was capable

of undergoing conformational maturationwithout functional BiP.

Finally, BiP was confirmed to be requiredfor translocation of both reporter proteins.The reporter protein Hsp150∆-NGFR

e was

labelled and chased at the restrictivetemperature and immunoprecipitated fromthe cell lysates and culture medium. Underthese conditions, the fusion protein wasfound in cell lysates as a faster migratingband as compared with the ER and matureforms (III, Figure 8). The fusion protein wasapparently on the cytoplasmic side of theER membrane, as it could not be labelledwith mannose. Thus, yeast BiP has twoseparate functions, assisting translocationand chaperoning.

29

DISCUSSION

1. Pretranslocational protein folding

Our studies demonstrated for the first timethat proteins can fold in the cytosol into anative-like conformation beforetranslocation into the ER. This is contraryto the previous view of the mechanism ofposttranslational translocation, wherecytoplasmic Hsp70 chaperones weresuggested to maintain the polypeptide in atranslocation-competent or unfolded formprior to translocation (Chirico et al., 1988;Deshaies et al., 1988). The Hsp150∆-β-lactamase fusion protein, destined forposttranslational translocation, was foldedinto a trypsin-resistant, active conformationin the yeast cytosol not only in translocation-defective mutants but also in wild-type cells.In the lumen of the ER, the reporter proteinwas correctly modified, and then secretedto the culture medium (Figure 4). Authenticβ-lactamase is a tight globular protein(Jelsch et al., 1992). Most of authenticHsp150 does not adopt any regularsecondary structure, with only the C-terminal domain, which is not present inHsp150∆-carrier, consisting of β-sheets, asdetermined by CD and NMR spectroscopy(Jämsä et al., 1995a). The structuraldifferences between Hsp150 and the fusionprotein likely explain the difference intranslocational retardation, which appearedto occur due to cytoplasmic eventsconcerning the β-lactamase portion. Thesimilarity of the Km values for both forms ofthe fusion protein on both sides of the ERmembrane to that of authentic E.coli β-lactamase indicated that they sharedsimilar structures at their activity sites.

Although we are first to report folding priorto translocation into the ER, as far as weknow, similar observations have been

made in studies of mitochondrialtranslocation. Folding of artificial reporterproteins and authentic mitochondrialcytochrome b2 prior to translocation intomitochondria have been reported (Glick etal ., 1993; Langer and Neupert, 1994;Wachter et al., 1994). In addition, Nguenet al . (1991) have observed the existenceof a protease-resistant form of a yeastsecretory protein prepro-α -factor on thecytoplasmic side of the ER membranewhen translocation was inhibited. Theysuggested that resistance occurred as aresult of aggregation or tight associationwith the ER membrane.

Disulfide bond formation of the β-lactamaseportion in the ER is essential for acquisitionof an active and secretion-competentconformation. The reducing agent DTTprevents proper folding, and the fusionprotein is retained in the ER (Simonen etal., 1994). However, in the cytoplasm, thefusion protein folded into an activeconformation even though the disulfidebond was not formed, reflecting thedif ferences of these compartments asfolding milieus. Authentic β-lactamase hasbeen shown to be active in the E.colicytosol, where disulfides are not formed(Plückthun and Knowles, 1987). Minsky etal. (1986) reported that after translocationacross the bacterial membrane β-lactamase occurs first as a trypsin-sensitive, membrane-bound form, which isthen converted to a trypsin-resistant andactive form, suggesting that the protein maybe unfolded and refolded aftertranslocation. Similarly, the dependence ofthe active conformation in the ER on thedisulfide bond suggests that Hsp150∆-β-

Discussion

30

lactamase unfolds at least to some extentprior to, during or after translocation intothe ER and refolds in the ER lumen. Inaddition, since the cysteines forming the

2. Unfolding precedes translocation

The crystal structure of authentic β-lactamase has a size of 32 x 37 x 53 Å(Jelsch et al., 1992). As the diameter of thetranslocation pore was reported to enlargeup to 60 Å when translocating a polypeptide(Hamman et al., 1997), and the sametranslocation channel was shown toaccommodate glycosylated incorrectlyfolded proteins on their way back to thecytosol (Pilon et al., 1997), the questionarose whether Hsp150∆-β-lactamase couldbe translocated in a folded or loosely foldedconformation. To answer this, two β-lactamase mutants were fused to theHsp150∆ fragment. One of these, E166A,irreversibly binds the antibiotic penicillin G(Adachi et al., 1991) and forms a stablecomplex (Strynadka et al ., 1992). Theadvantage of this mutant is its ability to lockthe β-lactamase into a folded structure,which is incapable of unfolding due to thecovalently bound antibiotic. The othermutant, E166D, binds the same antibioticin a reversible manner (Adachi et al., 1991)and confers reversibility to the stabilizationof the conformation.

To be active, proteins must fold into correctthree-dimensional structures. However,unfolding of proteins is also essential forseveral cell processes, such as ERtranslocation. β-lactamase, locked into afolded form due to the bound PenG, couldnot be translocated into the ER in livingyeast cells. The observation that the signalpeptide was uncleaved and that not eventhe first few of the multiple glycosylationsites of Hsp150∆ were occupied (Suntio etal., 1999), suggests that the fusion protein

disulfide bond are in the interior of the β-lactamase molecule (Jelsch et al., 1992),their oxidation would require at least partialunfolding of the protein.

did not penetrate into the translocationchannel. This was unexpected, since theHsp150∆ fragment has no structuralconstraints for translocation, as it occursmostly as a random coil (Jämsä et al.,1995a). It appears as though theapparently cytoplasmic unfoldingmachinery, the translocating substrate andthe translocation machinery, communicatewith each other, and that the translocationpore remains closed until completion ofunfolding. PenG and another β-lactamantibiotic cloxacillin, which bind reversiblyto E166D and native β -lactamase,respectively, did not prevent translocationof their respective fusion proteins. Thissuggests that the release of the drugallowed unfolding and subsequenttranslocation, and that prefolded proteinshave to unfold for translocation. Similarresults have been obtained inposttranslational translocation into themitochondria both in vitro (Eilers andSchatz, 1986) and in vivo (Wienhues et al.,1991). Translocation of a fusion proteinconsisting of a mitochondrial targetingsignal and mouse DHFR into isolated yeastmitochondria was prevented if DHFR wasbound to its ligand, methotrexate.Methotrexate was found to stabilize thestructure of DHFR, and thus wassuggested to block translocation throughmitochondrial membranes by preventingunfolding of the protein (Eiliers and Schatz,1986). Wienhues et al. (1991) reported asimilar translocation block in vivo. By usinga ligand of DHFR, which could be removedfrom the protein, they showed that thetranslocation block was reversible, further

31

supporting the necessity of unfolding fortranslocation. The import of foldedpreproteins into mitochondria can behundreds of times faster than theirspontaneous unfolding, indicating thatmitochondria can actively unfold proteins(Matouschek et al., 1997).

The Hsp70 protein of the ER lumen, BiP, isimportant in driving posttranslationaltranslocation of preproteins into the ER(Vogel et al., 1990). Hsp150∆-β-lactamasewas active on the cytoplasmic side of theER membrane in the absence of functionalBiP or its J-domain partner Sec63p.Translocation in these mutants wasblocked at a stage which precedes theunfolding step. According to the resultsobtained with sec61-41 cells, where theinsertion of preproteins into thetranslocation channel is prevented at therestrictive temperature, Hsp150∆-β-lactamase appeared to be unfolded to aninactive and trypsin- sensitive conformationwhen docked to the translocation channel,while the entire fusion protein was still onthe cytoplasmic side of the ER membane(Figure 4). In other words, the fusion proteinwas unfolded in the cytoplasm while boundto translocation channels, and before it hadbeen reached by lumenal signal peptidasecomplex and BiP. In a reconstituted systemwith purified translocation components inthe absence of a lipid bilayer, efficienttranslocation of prepro-α -factor wasachieved even if BiP was not present butsubstituted by antibodies against thetranslocating polypeptide (Matlack et al.,1999). However, folding of prepro-α-factorwas not studied, and it might not achieve afolded structure prior to translocation. Ourresults demonstrate that a prefolded proteinis unfolded in vivo while the entirepolypeptide resides on the cytoplasmic sideof the ER membrane. Thus, BiP does notactively pull and thereby unfold thetranslocating polypeptide. After unfolding

and translocation into the ER, Hsp150∆-β-lactamase was refolded in the ER andsecreted to the medium.

Despite translocation of proteins into theER and mitochondria sharing commonfeatures, the mechanisms of unfoldingseem to be dif ferent. The length of thepresequence of mitochodrial reporterproteins has been shown to affect theimport rate. Unfolding of a preprotein at the

Figure 4. Model for cytoplasmic folding andunfolding of Hsp150D-b-lactamase. The b-lactamase portion of the fusion protein folds toa native-like conformation in the yeast cytosolprior to translocation. Disulfide bond is notformed. When attached to the Sec61 complex,b-lactamase unfolds to inactive and proteasesensitive form even though the whole fusionprotein still resides on the cytosolic side of theER membrane. After translocation into the ER,the signal peptide is removed, the Hsp150D isO-glycosylated, the disulfide bond is formed,and the protein refolds to a conformation withsimilar catalytic properties as the cytosolic form.

Discussion

32

mitochondrial surface was acceleratedwhen its presequence was sufficiently longto span both mitochondrial membranes andto interact with Hsp70 of the mitochondrialmatrix (Matouschek et al ., 1997). Theunfolding pathway of a mitochondrialbarnase preprotein was distinct from itsspontaneous unfolding pathway (Huang etal., 1999). Just like many enzymes catalyzechemical reactions by changing reactionpathways, the protein import machinerycatalyzes protein unfolding by changing theunfolding pathway of the substrate protein.Temperature-sensitive mutants ofmitochondrial matrix Hsp70, which lead toits inactivation, inhibit unfolding,translocation and subsequent folding ofmitochondrial precursors inside themitochondria (Gambill et al., 1993; Vooset al., 1993). Mitochondrial Hsp70s are themajor consumers of ATP duringtranslocation (Glick et al ., 1993). Theyseem to function as active motors togetherwith Tim44, a mitochondrial innermembrane protein, by pulling the precursoracross the membrane (Voisine et al., 1999).The N-terminal amino acids of an F

0 –

ATPase subunit were translocated, andonly the fusion partner, DHFR, stabilizedby methotrexate, was exposed to thecytoplasmic side of the mitochondrialmembrane (Voisine et al ., 1999). Import ofloosely folded precursors have beenreported to occur in the absence offunctional Tim44 (Bömer et al., 1998; Merlinet al., 1999), but a stably folded preproteinmust be actively pulled and thus unfolded,and not only trapped, by matrix Hsp70s(Bömer et al., 1998; Merlin et al ., 1999;Voisine et al., 1999). Thus, in mitochondria,matrix Hsp70 unfolds preproteins fortranslocation.

As Hsp150∆-β-lactamase was almostentirely exposed to the cytosol duringunfolding, the unfolding machinery must becytosolic. The cytoplasmic reporter protein

interacted with cytoplasmic Hsp70chaperones as shown by coimmuno-precipitation experiments, and the ER forminteracted with the lumenal Hsp70, BiP(Jämsä et al .,1995). In spite of the highhomology of these proteins, they cannotsubstitute for each other in translocationacross the ER membrane (Brodsky et al.,1993). Hsp150∆-β-lactamase existed in afolded conformation in the cytoplasm priorto its translocation, even though it was inassociation with cytoplasmic Hsp70proteins. This could perhaps indicate a rolefor Ssa proteins in unfolding ofpretranslocationally folded secretoryproteins. Translocation of pre-pro-α-factorand folding of nascent luciferase have beendemonstrated in vitro that to beindependent of the functional Ssa1 andSsa2 proteins, whereas the refolding ofdenatured luciferase requires the presenceof these chaperones (Bush and Meyer,1996). Ssa proteins were thus proposed tounfold the translocation-incompetentstructures in the yeast cytosol beforetranslocation.

β-lactamase must unfold for translocation,but to what extent it unfolds remains to bestudied. The folding pathway of mostproteins in vitro contains a partially foldedintermediate known as the molten globule.Proteins have been postulated to be in themolten globule state during translocationacross membranes (Bychkova et al., 1988).By introducing disulfide bridges thatcovalently linked two to five β-sheetstogether, Schwartz et al . (1999)demonstrated in vitro that preproteins arenormally fully unfolded during translocationinto mitochondria, but residual structurescan be imported, although less efficiently.The diameter of the protein conductingchannel of the outer mitochondrialmembrane reconstituted in lipid vesicleswas measured to be 20 Å (Künkele et al.,1998), thus sufficient to accommodate

33

secondary structural elements. It is not yetknown whether the channel enlarges duringtranslocation. Nor can it be ruled out that

3. The functions of BiP in translocation and folding are distinct

Since unfolding of pretranslocationallyfolded proteins precedes translocation, theproteins must refold in the ER lumen. Theinvolvement of BiP/Kar2p in translocationas well as in facilitating folding complicatesin vivo studies on its role in folding. Theuse of DTT to prevent folding oftranslocated proteins helped to define thetwo functions of BiP, and made it possibleto study its role in folding in vivo. The tworeporter proteins used in this study, E. coliβ-lactamase and the ectodomain of ratnerve growth factor receptor (NGFR

e),

have different three-dimensionalstructures, which likely explains theirdifferent requirements of BiP inconformational maturation after reducingconditions. DTT treatment led to theretention of both reporter proteins in the ER.This has also been shown to occur inmammalian cells, where reduced VSV Gprotein was retained in the ER, as revealedby cell fractionation and morphologicalstudies (Hammond and Helenius, 1994),and in yeast, as DTT-reduced CPY lackedextended glycan structures, indicating itslocalization in the pre-Golgi compartment(Holst et al., 1996). When retained underreduced conditions in the ER, the NGFR

eportion of the fusion protein was moreefficiently N-glycosylated than in normalconditions, where the N-glycosylation siteis variably occupied (Holkeri et al ., 1996;Simonen et al., 1996). Thus, folding andglycosylation seem to compete with eachother, and folding of polypeptides prior toglycosylation hides the potentialglycosylation site, thereby preventingglycosylation. The reducing conditions keepthe N-glycosylation site of NGFR

e available

mitochondrial membranes might contain arange of import channels of different sizes.

for the oligosaccharyltransferase over aprolonged period, leading to an increasedlevel of modification.

Hsp150∆-NGFRe folded to an apparently

correct three-dimensional structure andwas allowed to leave the ER to be secretedin conditions where BiP was not functioningand could not facilitate folding. As amultidomain protein, NGFR

e probably folds

domain by domain when emerging from thetranslocation channel (Netzer and Hartl,1997). This decreases the enormousnumber ( 3.16 x 1011) of possible disulfidebonds formed by 24 cysteines of NGFR

e to

15. In human TNF receptor (TNFR), thecysteines seem to form disulfides insequence with the first possible freecysteine. Furthermore, in the case ofTNFR, the cysteine residues were notcritical to folding (Banner et al., 1993). Thisindicates that perhaps the folding of NGFR

eis not severely complicated in reducedconditions; the protein is able to acquire anearly correct conformation even in theabsence of disulfides, which would quicklybe formed if oxidizing conditions wereresumed in the ER. This would explain whythe protein is able to mature to a secretion-competent conformation so rapidly.

As a globular protein, β-lactamase probablyneeds to be fully translocated into the ERbefore the final structure can be attained.The disulfide bond resides in the middle ofthe molecule and its formation presumablyrequires that the surrounding areas arekept unfolded or loosely folded. BiP bindsto unfolded nascent polypeptide sequences(Simons et al ., 1995; Hendershot et al .,

Discussion

34

1996).This could explain the requirementof BiP for folding of β-lactamase. BiP couldbe coimmunoprecipitated with Hsp150∆-β-lactamase (Jämsä et al., 1995b), whereasno interaction between Hsp150∆-NGFR

eand BiP was detected (data not shown). Inkar2-159 cells, the ATPase activity of BiPis defective (Vogel et al., 1990), leading tostable binding of BiP to its substrates(Palleros et al., 1993). Mutant BiP was likelyto be bound permanently to reduced β-lactamase, thereby preventing folding,supporting the view that BiP would favourfolding by preventing aggregation.

Taken together, we have shown thatfacilitation of translocation and folding aretwo separate functions of BiP. Furthermore,although BiP is required for

posttranslational translocation of allsecretory proteins studied thus far, itsfunction in folding is substrate-specific.Proteins that easily fold into nativeconformations perhaps do not need to beaided by BiP. Differences in requirementsof BiP for posttranslocational chaperoninghave been reported in mammalian cells(Pittman et al., 1994; Morris et al., 1997).Recently, Hellman et al. (1999) showed thatthe rate and stability of protein foldingdetermines whether or not a particular siteis recognized by BiP. BiP was found to bindpreferentially to slowly folding or unstableproteins.

The results of this study show for the firsttime that in living S. cerevisiae cellsposttranslationally translocated proteinscan fold into native-like conformations onthe cytosolic face of the ER membrane.When attached to the translocon, prefoldedproteins unfold for translocation. Theunfolding step occurs before initiation ofpenetration through the translocation pore,

thus revealing a novel function in the yeastcytosol, that of unfolding of prefoldedproteins. In the ER, translocated proteinsrefold into secretion-competentconformation. Even though chaperone BiPis required for translocation of all proteinsubstrates, its requirement forconformational maturation was found to beprotein-specific.

CONCLUSIONS

35

ACKNOWLEDGEMENTS

This work was carried out at the Instituteof Biotechnology, with the financial supportof the Graduate School in Microbiology andthe Viikki Graduate School in Biosciences.

I wish to express my gratitute to mysupervisor, Professor Marja Makarow, forthe oportunity to work in her group. Herenthusiasm, positive attitude andcontinuous interest made working in theyeast group a pleasure.

I thank Professor Mart Saarma, the headof the Institute of Biotechnology, forproviding excellent working facilities andcreating a dynamic, stimulating and warmworking environment. Professor LeeviKääriäinen I thank for the detailed timetableregarding my dissertation. I thank alsoprofessor Timo Korhonen, the head of theGraduate School in Microbiology. I amgrateful to Docent Pekka Lappalainen andDr. Vesa Kontinen for their critical readingof the manuscript. I sincerely thank themfor pointing out the sections of the textwhich were probably written after falling intoa deep sleep. Heli Ruoho is acknowledgedfor the layout.

I warmly thank all the former and presentmembers of the yeast group for the lively,lovely and loud atmosphere at work. Ourtea breaks, all day long with a good laugh,made brighter even the gloomiestmoments. I will never forget the mostenjoyable parties both at work and inprivate, they really shook me all night long!

I especially wish to thank Marjo Simonenand Eija Jokitalo for the very nice welcometo the yeast group and the advice in thebeginning, and Eija for her friendship eversince and the criticism without mercy notonly at work but also at shopping. I thankAnna Liisa Nyfors for the excellent technicalhelp, patience and her good eyes in findinglost bottles and tubes. To Heidi, Nina, Mari,Netta, Eeva, Taina, Ansku, Maria, Lauraand Leena I owe my thanks for friendship,helpful hands in every day problems andshaping activities outside of work. Theboys, Vijay, Anton, Juha and Qiao, in spiteof trying hard to not to be different than therest of us, reaching a climax when theydressed up as women, have provided awelcome masculinity to the traditionallyquite feminine appearance of the yeastgroup. I also thank Qiao for the efficientcloning work in the last article.

I thank my parents and relatives for theirlove and support even though I neverbecame a lawyer. All my friends arethanked for the relaxing time together. I owemy special thanks to Hanna for the long-distance calls whenever I needed a shrink!My deepest thanks go to Olli and Topi, fortheir love and trust and more precisely, Ollifor creating order and Topi for creatingrefreshing disorder in the study. Topi, yourtraining of my skills in debate with adetermination of a three-year old isacknowledged!Helsinki, August 2000

Acknowledgements

36

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