Reviews: Parasite cell Biology: 1 The ﬂagellum and ... flagellum.pdf · including Trypanosoma brucei, Trypanosoma cruzi, Leishmania species, Crithidia fasciculata and others has

Molecular & Biochemical Parasitology 115 (2001) 1–17

Reviews: Parasite cell Biology: 1

The flagellum and flagellar pocket of trypanosomatids

Scott M. Landfear *, Marina IgnatushchenkoDepartment of Molecular Microbiology and Immunology, Oregon Health Sciences Uni�ersity, Portland, OR 97201, USA

Received 9 November 2000; received in revised form 26 January 2001; accepted 5 March 2001

Abstract

The flagellum and flagellar pocket are distinctive organelles present among all of the trypanosomatid protozoa. Currently,recognized functions for these organelles include generation of motility for the flagellum and dedicated secretory and endocyticactivities for the flagellar pocket. The flagellar and flagellar pocket membranes have long been recognized as morphologicallyseparate domains that are component parts of the plasma membrane that surrounds the entire cell. The structural and functionalspecialization of these two membranes has now been underscored by the identification of multiple proteins that are targetedselectively to each of these domains, and non-membrane proteins have also been identified that are targeted to the internal luminaof these organelles. Investigations on the functions of these organelle-specific proteins should continue to shed light on the uniquebiological activities of the flagellum and flagellar pocket. In addition, work has begun on identifying signals or modifications ofthese proteins that direct their targeting to the correct subcellular location. Future endeavors should further refine our knowledgeof targeting signals and begin to dissect the molecular machinery involved in transporting and retaining each polypeptide at itsdesignated cellular address. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Trypanosomatid protozoa; Flagellum; Flagellar Pocket; Organelle-specific proteins; Review

www.parasitology-online.com.

1. Introduction

Two distinctive features in the cell biology of try-panosomatid protozoa are the presence of flagella in atleast some life cycle stages and the existence of aprominent invagination of the plasma membrane calledthe flagellar pocket [1]. The purpose of this review is toprovide an overview of recent as well as some longerstanding discoveries concerning the nature of these twoclosely apposed organelles. This article is organizedlargely around specific proteins that have been shownto reside in either the flagellum or the flagellar pocket.The reason for this approach is that a number ofexcellent reviews already exist [2–6] that deal wholely

or partially with the more general cell biology of theflagellar pocket and flagellum. In contrast, recentlypublished material has increased the number of proteinsknown to reside within the membranes or lumina of theflagellum or flagellar pocket and has given us novelmolecular markers with which to probe the biologicalfunctions of these organelles.

The surface membrane of kinetoplastid protozoa,including Trypanosoma brucei, Trypanosoma cruzi,Leishmania species, Crithidia fasciculata and others hasbeen divided into three morphologically distinct subdo-mains [2]: the flagellar membrane, the flagellar pocket,and the pellicular plasma membrane (Fig. 1). It is nowrecognized that each of these domains represents a

Abbre�iations: BSA, Bovine serum albumen; CRAM, cysteine-rich acidic integral membrane protein; ER, endoplasmic reticulum; ESAG,expression site associated gene; FCaBP, flagellar calcium binding protein; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol;HDL, high density lipoprotein; HRP, horse radish peroxidase; IFT, intraflagellar transport; LDL, low density lipoprotein; LPG, lipophosphogly-can; MVT, multivesicular tubule; PFR, paraflagellar rod; PPG, proteophosphoglycan; sAP, soluble acid phosphatase; SDS, sodium dodecylsul-fate; Tf, transferrin; TFBP, transferrin binding protein; TLTF, T lymphocyte triggering factor; VSG, variant surface glycoprotein.

* Corresponding author. Tel.: +1-503-4942426; fax: +1-503-4946862.E-mail address: [email protected] (S.M. Landfear).

0166-6851/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 6 -6851 (01 )00262 -6

S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–172

Fig. 1. Subcellular structure of a bloodstream form African trypanosome (reproduced from [5] by copyright permission of Elsevier Science Ltd.).The abbreviations are: az, adhesion zone at the entrance of the flagellar pocket; cv, coated vesicles; er, endoplasmic reticulum; fl, flagellum; fp,flagellar pocket; gl, glycosome, a membrane bound organelle involved in glycolysis and other metabolic pathways; go, Golgi apparatus; k,kinetoplast containing highly catenated kinetoplast DNA; l, lysosome; m, mitochondrion; mt, subpellicular microtubules; n, nucleus, sc, surfacecoat containing variant surface glycoprotein; tv, tubulovesicular structure. The length of the cell is approximately 20 �m. The cell surfacemembrane can be divided into the pellicular plasma membrane surrounding the cell body, the flagellar pocket membrane, and the flagellarmembrane.

highly specialized membrane with distinctive functionsand unique protein and possibly lipid compositions.Thus, the pellicular plasma membrane surrounds thebody of the cell and is attached to a dense corset ofhighly stable, cross-linked microtubules. This mem-brane contains many of the permeases that mediateuptake of nutrients via classical transporter cycles, itprovides the cell body with its shape, and in somecases, it is densely covered with a protein or glycol-ipid coat that protects the parasite against host im-mune responses, as in the case of the variant surfaceglycoproteins (VSGs) of T. brucei [7] and the abun-dant glycolipid lipophosphoglycan (LPG) of Leishma-nia species [8]. The flagellum (Fig. 2) is the classicalmotility organelle that moves the parasite forward bywave-like beats of the microtubule-based flagellar ax-oneme, but it is also involved in additional biological

activities such as the attachment of parasites to theendothelium of their insect hosts [1], and it may alsobe a specialized sensory organelle. The flagellarpocket, a deep invagination at the base of the flagel-lum (Fig. 3) is responsible for uptake of larger nutri-ents via receptor-mediated endocytosis, for secretionof proteins into the extracellular medium, and for in-tegration of membrane proteins into the cell surface.It is noteworthy that these three membranes are phys-ically contiguous, and all constitute part of theplasma membrane despite their highly differentiatedbiological functions. The identification of proteinsthat are localized discretely to one of these plasmamembrane components has helped to delineate thedistinct functions of each of these membrane surfaces.We are now beginning to understand how each com-partment is maintained as a unique entity by identify-

Fig. 2. The cytoskeleton of the flagellum and paraflagellar rod of L. mexicana (reproduced from [44], by copyright permission of Elsevier ScienceLtd.). (A) A whole-mount negatively stained cytoskeleton is shown, including the basal body ‘b’ where the flagellum initiates, the flagellar axoneme‘a’ which is the microtubule-based structure that generates flagellar motility, and the fibrous paraflagellar rod ‘p’ that runs adjacent to theaxoneme. The subpellicular microtubules that lie underneath the pellicular plasma membrane can be seen in the cell body region at the left of thefigure. (B) A transverse view of the flagellar cytoskeleton showing the ‘9+2’ arrangement of singlet and doublet axonemal microtubules. Thefibrous paraflagellar rod is underneath the axoneme and consists of the proximal, intermediate, and distal domains (labeled ‘pd’, ‘id’, and ‘dd’,respectively).

S.M. Landfear, M. Ignatushchenko / Molecular & Biochemical Parasitology 115 (2001) 1–17 3

Fig. 3. A thin section electron micrograph through the flagellarpocket of a bloodstream form T. brucei parasite showing the contigu-ous pellicular plasma membrane, flagellar pocket membrane, andflagellar membrane (reproduced from [6] by copyright permission ofElsevier Science Ltd.). The flagellum initiates at the basal body nearthe base of the flagellar pocket and extends through the anterioropening of the pocket at the top of the figure. An electron denseregion where the surface membrane apposes the flagellar membraneconstitutes the ‘junctional complex’ or ‘adhesion zone’. The lumen ofthe flagellar pocket is filled with diffuse electron dense material. Thesolid arrow indicates a cytoplasmic vesicle adjacent to the flagellarpocket, and the open arrow indicates the kinetoplast DNA that isenclosed by the mitochondrial membrane and is immediately poste-rior to the basal body. Subpellicular microtubules can be seen under-neath the pellicular plasma membrane at the top and bottom of thefigure, where they have been sectioned at a glancing angle. The scalebar represents 0.5 �m.

rest of the surface bilayer is the observation that severalwell-characterized membrane proteins in varioustrypanosomatids are localized discretely in the mem-brane of this organelle. A number of Ca+2-bindingproteins of both T. cruzi and T. brucei, receptor--adenylate cyclases of T. brucei, and a glucosetransporter isoform in L. enriettii are all present in theflagellar membrane, while they occur at low orundetectable levels on the pellicular plasma membrane.In addition, earlier biochemical studies [9] detectedspecific proteins on SDS-polyacrylamide gels using amembrane fraction from purified flagella, although thisfraction was not directly compared with the pellicularplasma membrane to determine which of these proteinswas truly flagellar-specific. Furthermore, studies on thelipid composition of flagellar membranes of severalmicro-organisms [10] have revealed high sterol/phospholipid ratios compared with other membranes,and this pattern of differential lipid content appears toapply in trypanosomatids, as well, where the sterol-binding antibiotic filipin intercalates to a high degree inthe flagellar membrane [9]. A question of centralimportance is to determine how proteins are selectivelytargeted to or excluded from the flagellar membrane,and studies on several of the flagellar membraneproteins are beginning to elucidate this process.Another intriguing question has to do with thebiological significance of flagellar localization; that is,what are the specific functions of proteins that resideexclusively or preferentially in this membrane?

2.1. Flagellar Ca+2-binding proteins

Over 10 years ago, Engman et al. [11] identified anEF-hand flagellar Ca+2-binding protein, FCaBP, fromT. cruzi. Subsequent studies revealed that FCaBP wasdually modified with myristate on the amino group ofthe NH2-terminal glycine and palmitate on the thiolgroup of the cysteine at residue 3, and that both ofthese acyl groups were required for membrane associa-tion and for localization to the flagellum [12]. Thisprotein appears to be associated with the cytosolic faceof the plasma membrane and does not contain anyapparent transmembrane segments. Furthermore, a fu-sion protein between the first 24 amino acids of FCaBPand Green Fluorescent Protein (GFP) was also targetedto the flagellar membrane, indicating that all of theessential targeting information was contained withinthis NH2-terminal segment of the protein. It is currentlynot clear whether the dual acylation that mediatesmembrane association is itself responsible for the dis-crete flagellar localization, via lipid sorting, or whethera targeting sequence located within the first 24 aminoacids is required for restriction to this organelle. FCaBPhas been shown to associate with the flagellar mem-brane in a Ca+2-dependent manner. This property is

ing targeting signals that sort proteins to each of thesedifferentiated membranes or to the structures enclosedby these membranes.

2. Flagellar membrane

The most definitive evidence that the flagellarmembrane is distinct in protein composition from the


reminiscent of the mammalian protein recoverin [13],another EF-hand Ca+2-binding protein from retinal rodcells that associates with the plasma membrane, whenintracellular Ca+2 levels rise. Membrane association ofrecoverin leads to inhibition of rhodopsin kinase andthus serves to transmit changes in Ca+2 levels into abiological readout [14]. The observations in T. cruzireveal a conserved mechanism of regulated membraneassociation and suggest by analogy that FCaBP is verylikely to be involved in signal transduction mediated bychanges in intracellular Ca+2 levels. However, the molec-ular component of this signaling pathway that is down-stream of FCaBP has not yet been identified.

A family of related EF-hand Ca+2-binding proteinshas also been identified in T. brucei and shown to localizeto the flagellum [15,16]. Hence, these flagellar associatedproteins may be involved in regulating a variety ofcellular processes in various trypanosomatids.

2.2. Receptor-adenylate cyclases

T. brucei [17] and L. dono�ani [18] express a family ofadenylate cyclases that appear to have a large extracellu-lar domain, a single transmembrane domain, and anintracellular adenylate cyclase domain. These proteinsare likely to serve some function as signal transductionreceptors and are structurally related to the well-charac-terized mammalian atrial natriuretic peptide receptorthat is a ligand-activated guanylate cyclase [19]. Pays andcolleagues [17] demonstrated by both light and electronmicroscopy that a member of this family, ESAG 4, is

localized exclusively to the flagellar membrane, althoughwhat portion of the protein is involved in the organelle-specific trafficking is not clear. To date, neither theligands for these putative receptors nor the probable rolesof the receptors in signal transduction have been iden-tified.

2.3. Flagellar glucose transporter

Studies on glucose transporters from L. enriettii iden-tified two isoforms, ISO1 and ISO2, that differ exclu-sively in the NH2-terminal hydrophilic domain that isthought to be located on the cytoplasmic side of thesurface membrane [20]. Localization of these two iso-forms by confocal immunofluorescence and immunoelec-tron microscopy revealed that ISO1 is restricted to theflagellar membrane (Fig. 4), whereas ISO2 is located inthe pellicular plasma membrane but not the flagellarmembrane [21]. Furthermore, a chimera between theunique NH2-terminal domain of ISO1 and anotherpellicular plasma membrane glucose transporter, D2,trafficked to the flagellar membrane, demonstrating thatthe ISO1 domain was sufficient for flagellar targeting[22]. Subsequent deletion and site-directed mutagenesisidentified a sequence of five contiguous amino acids,RTGTT [23] located between positions 25–29 of theISO1 sequence, that was central in flagellar targeting.Thus, a deletion of 20 amino acids from ISO1 did notinterfere with flagellar targeting, whereas fusing the first35 amino acids of ISO1 onto ISO2 resulted in flagellarlocalization of the chimera, together showing that se-

Fig. 4. Flagellar localization of the glucose transporter ISO1 in L. mexicana. Parasites were transfected with a construct that expresses EnhancedGreen Fluorescent Protein (EGFP) [103] fused onto the COOH-terminus of ISO1. Cells were fixed with methanol and stained with an anti-�-tubulinantibody followed by anti-IgG antiserum conjugated to Texas Red and examined by fluorescence microscopy. (A) ISO1-EGFP fusion showingstaining (green) on the flagellum and flagellar pocket. (B) �-tubulin staining (red) of the cell body and flagellum. (C) Merged images of (A) and(B).


quence between positions 20–35 contained a flagellartargeting sequence. Alanine scanning mutagenesis ofISO1 showed that the G27A, T28A, and T29A mutantstrafficked less efficiently than wild type transporter tothe flagellar membrane, whereas R25A and T26Atrafficked more efficiently to the flagellum and wererelatively less abundant in the flagellar pocket than thewild type ISO1. However, addition of the RTGTTsequence to ISO2 was not by itself sufficient forflagellar targeting, indicating that the structural contextof this sequence is important for function. Thesemutagenesis studies have identified a sequence withinISO1 that may interact with other components of aflagellar targeting machinery, and future efforts todetect potential associations of ISO1 with otherproteins may begin to dissect the targeting pathway.

2.4. GP72, a protein in�ol�ed in flagellar attachment

Null mutants of the gp72 gene of T. cruzi, whichencodes an immunodominant membrane glycoprotein,revealed a remarkable phenotype: the flagellum was nolonger attached to the cell body after it emerged fromthe flagellar pocket, and the motility of the mutants wasreduced [24]. Localization of epitope tagged Gp72revealed that this protein is distributed over the cellbody surface and the flagellar pocket, but that it isconcentrated in the proximal region of the flagellumand is undetectable at the distal tip of the flagellum[25]. Thus, despite its flagellar phenotype, this protein isquite widely distributed over the parasite surface. It ispossible that Gp72 interacts with the flagellar adhesionzone, a region where the flagellar membrane adheres tothe pellicular plasma membrane [2], and that theabsence of this interaction could explain themorphology of gp72 null mutants. Of additionalinterest, gp72 null mutants have greatly reduced abilityto establish infections in the insect vector Triatomainfestans [26].

A homolog of gp72 has been identified in T. bruceiand is designated the fla1 gene [27]. The Fla1 protein isevenly distributed along the flagellum, except at theproximal end where the region of the flagellar pocketstains more strongly. This distribution is similar to thatof a 88 kDa membrane protein from T. brucei that wasearlier observed to concentrate in the flagellarattachment region [28]. However, unlike Gp72, it wasnot possible to directly assess the function of Fla1, asnull mutants could not be obtained and apparentlywere not viable.

2.5. Mechanism of targeting to the flagellar membrane

How are proteins like FCaBP, ESAG 4 and ISO1selectively targeted to the flagellar membrane? In princi-ple, they could either be actively sequestered in this

specialized membrane, or they could diffuse into theflagellum and then be held in the organelle by retention.Alternatively, the acyl modifications on some proteinssuch as FCaBP could cause partitioning into the uniquelipid environment of the flagellar membrane. Further-more, other proteins like ISO2 are not present in theflagellar membrane and could be either actively ex-cluded from this compartment or retained over thepellicular plasma membrane. The answers to thesequestions are not yet clear, but some relevant data areavailable. Thus ISO1 in which the first 30 amino acidshave been deleted traffics to the pellicular plasma mem-brane [22], suggesting that this route may be a defaultpathway, that operates if no flagellar targeting signal ispresent.

Since all surface membrane proteins are thought toenter the flagellar pocket membrane first [5,6,29], andsince both the flagellar ISO1 and the pellicular plasmamembrane ISO2 are present in the flagellar pocketmembrane, it is likely that this latter subdomain is thesite for differential sorting of flagellar and pellicularplasma membrane proteins. Presumably, a dominantflagellar targeting signal causes routing of proteins intothe flagellar compartment, whereas polypeptides thatdo not contain this signal may traffic by default to themembrane surrounding the cell body.

While the machinery responsible for flagellar mem-brane targeting has not been identified, studies onflagellar assembly in the green alga Chlamydomonasreinhardtii may offer some clues about how this processmight occur. An activity designated ‘intraflagellartransport’ (IFT) [30,31] is responsible for the movementof newly synthesized components of the flagellar axon-eme in both anterograde and retrograde directions.Large complexes termed ‘rafts’ that contain at least 15proteins are moved in both directions beneath theflagellar membrane. Furthermore, the anterogrademovement has been shown to be dependent upon akinesin-like protein FLA10, and retrograde movementis dependent upon a dynein-like protein DHC1b.Hence, there is a molecular machinery dedicated tomoving complex particles along the flagellum. Althoughmost of these studies have analyzed movements ofnon-membrane proteins, other work has also identifiedan apparently related motility process that moves com-ponents associated with the flagellar membrane [32,33],and like IFT, this movement of membrane componentsis also inhibited by mutations in the fla10 gene [31,34].Hence, it is likely that some flagellar membraneproteins are directed to their correct location by IFT ora similar process. It is intriguing that three point muta-tions in the ISO1 flagellar targeting domain resulted ina phenotype that could be explained by impairedanterograde transport, whereas two other point mu-tants had a phenotype reminiscent of impaired retro-grade transport [23].


2.6. Is the flagellum a specialized sensory organelle intrypanosomatids?

Evidence from other systems representing both themicrobial and mammalian world has highlighted theinvolvement of cilia and flagella in sensing the environ-ment. Until recently, this latter function has been lesswell appreciated than the better-established role ofthese organelles in motility. Thus signal transductionreceptors that mediate cellular responses associatedwith mating of C. reinhardtii gametes are located spe-cifically in the flagellar membrane [35]. The complexfamily of olfactory receptors in mammals that mediatethe sensation of smell are localized to the ciliary mem-branes of olfactory neurons [36], and odorant receptorsin Caenorhabditis elegans are also localized to olfactorycilia [37]. Furthermore, FCaBP and the receptor adeny-late cyclases of trypanosomes are very likely involved insignal transduction and may mediate a flagellar-specificsensory function. Although the particular function ofthe flagellar glucose transporter ISO1 is currently un-known, the recent observations that glucose trans-porter-like proteins in yeast [38] and other bonafidetransporters in various other micro-organisms andplants [39–42], as well as the human GLUT1 glucosetransporter [43] can function as signal transductionreceptors to monitor the level of their ligands in theenvironment raises the intriguing possibility that thisflagellar isoform might be involved in glucose sensing.The flagellar localization of various sensory membraneproteins might be necessary for interaction with down-stream components of signaling pathways that couldthemselves be localized to the interior of the flagellum.Further studies on the biological functions of flagellarmembrane proteins should help to elucidate the likelysensory functions of this organelle.

3. Paraflagellar rod

While trypanosomatids have many conventionalstructural components of flagella [4], they also containan unusual fibrous body called the paraflagellar rod(PFR) that is constituted from discrete filaments, runsalong the length of the flagellum, and is attached to theflagellar axoneme [44] (Fig. 2). In T. brucei, the majorcomponents of the PFR are two closely related proteinsdesignated PFR-A and PFR-C, each encoded by acluster of 4 repeated genes [45]. Similar genes andproteins have been identified in L. mexicana and T.cruzi, where they are called PFR-2, PFR-1 and PAR-2,PAR-3, respectively [4]. This structure, which also oc-curs in euglenoids, had been speculated to play a role inmotility, but definitive evidence for such a function wasnot available until recently, with the development intrypanosomatids of gene knockout technology using

homologous gene replacement or RNA interferencemethods.

A PRF-2 null mutant was generated by homologousgene replacement in L. mexicana [46]. The mutantparasites assembled a residual PFR that stained withantisera against PFR-1, revealing that the PFR isgreatly altered but probably not completely disruptedin this mutant. Notably, the null mutants had anapproximately 4-fold reduced velocity of forward motil-ity and an altered flagellar beat pattern exhibiting areduced wavelength and somewhat lower beat fre-quency. This motility phenotype might be explained byreduced elastic bending resistance resulting from alter-ation of the PFR. In T. brucei, the PFR-A mRNA wasablated by RNA interference [47] using overexpressionof antisense RNA from an expression construct inte-grated into the PFR-A gene locus [48]. These functionalnull mutants had an even more dramatic phenotypethan the L. mexicana PFR-2 null mutants, as they werelargely paralyzed and sedimented to the bottom of thetissue culture flask during growth. The PFR appearedto be disrupted, and PRF-C protein was released intothe detergent soluble fraction, in contrast to its locationin wild type parasites. Together, these two manuscriptsprovide the first definitive evidence that the PFR has acentral role in motility of trypanosomatids. A furtherunderstanding of the details of PFR ultrastructure, thebiophysical properties of this rod, and the identity andfunction of less abundant PFR proteins [4] shouldfurther clarify the function of this remarkable subcellu-lar structure.

An intriguing study by Gull and colleagues [49] hasidentified a segment of the PFR-A protein of T. bruceithat is necessary for targeting to the paraflagellar rod.Deletion mutagenesis revealed that a region betweenamino acids 514 and 570 of this 600 amino acid proteinwas essential for addition of PFR-A to the paraflagellarrod, but this sequence itself was not sufficient to targetGFP to this fibrous structure. Furthermore, the se-quence between amino acids 559 and 574 showed sub-stantial homology to similar regions in paraflagellar rodproteins from other kinetoplastids and from Euglenagracilis as well as to a sequence in the heavy chain of anaxonemal dynein from C. reinhardtii, implying that acommon signal for flagellar targeting or assembly maybe conserved across these species. More recent studiesrevealed that the tripeptide HLA, located at aminoacids 563–565, is shared in common with a novelactin-related protein, TrypARP, that is also targeted tothe paraflagellar rod and that deletion of this tripeptidefrom either PFR-A or TrypARP prevents assembly ofthe mutant protein into the rod [50], thus identifying aprobable paraflagellar rod assembly motif. In addition,PFR-A appeared to have a major site of addition at thedistal tip of the paraflagellar rod but to undergoturnover along the length of the rod.


4. Flagellar pocket

The flagellar pocket is a deep invagination of theplasma membrane that is located at the base of theflagellum (Figs. 1 and 3). Although the pocket has asomewhat distinct morphology in different kinetoplas-tids [1], its functions are thought to be similar in allcases. This pocket is located at the anterior end of thecell, it encloses the base of the flagellum, and it appearsto be surrounded at its opening by a desmosome-likethickening [3] variously referred to as the ‘junctionalcomplex’ [2], the ‘zone of adhesion’ [5], or the ‘maculaeadherens’ [6] (Fig. 3). It has been proposed but notdemonstrated that this junctional complex may restrictthe flow of material into and out of the flagellar pocket,but macromolecules can clearly move both into and outof the pocket (see below).

It has been appreciated for some time that the flagel-lar pocket membrane, representing between 0.4 and 3%of the cell surface [2,6], is highly specialized and is theonly known site for endocytosis, for secretion ofproteins from the cell, and for addition of integralmembrane proteins to the cell surface [5,6,29]. Sincethese vesicle-dependent events are restricted to a verysmall component of the surface membrane yet blood-stream trypanosomes can internalize a membrane areaequivalent to that of the flagellar pocket approximatelyevery 2 min [51], the flagellar pocket is probably themost active organelle known for endocytosis [5]. Whilea dense network of subpellicular microtubules is at-tached to the cytoplasmic side of the pellicular plasmamembrane [4] (Figs. 2 and 3), these microtubules areabsent from the flagellar pocket, with the exception ofa quartet of specialized microtubules that run along onesurface of the flagellar pocket [6]. It is thought that thesubpellicular microtubule network may prevent pro-cesses such as vesicle fusion and receptor-mediatedendocytosis from occurring at any place outside theflagellar pocket, but the molecular mechanisms thatrestrict these events to the flagellar pocket are notknown and could well be more complex than simplephysical exclusion by the cytoskeletal network. In con-trast, invaginations resembling the coated pits of mam-malian cells have been observed by various groups [5]in the membrane of the flagellar pocket. In addition,studies on uptake of several ligands have revealed thathorseradish peroxidase (HRP), ferritin, and BSA-goldare taken up by non-saturable fluid-phase endocytosisthat is relatively slow [51], whereas transferrin (Tf) andlow density lipoproteins (LDL) are endocytosed by arapid, saturable, and ligand-specific process similar toreceptor-mediated endocytosis in mammalian cells.Both types of endocytic process appear to be restrictedto the flagellar pocket membrane. Furthermore, al-though only examined for a few cases, both membranebound [29] and secreted proteins [52] appear to reach

the cell surface at the flagellar pocket membrane, un-derscoring the role of this membrane in delivery ofproteins to the cell surface and exterior. Finally, thelumen of the flagellar pocket can be seen to containelectron dense material (Fig. 3) and clearly harborsspecific proteins [52] that are secreted into this space.All of these observations support the conclusion thatthe flagellar pocket is a highly differentiated componentof the plasma membrane that is specialized for uptakeand secretion of molecules required by or released fromthe parasite.

Although an exhaustive review of secretion and en-docytosis in trypanosomatids is beyond the scope of thecurrent article, Sections 4.1and 4.2 below provide anoverview of these processes as they relate to the flagel-lar pocket. Subsequent sections review specific proteinsthat are known to be components of the membrane orthe lumen of the flagellar pocket. The identification andfunctional investigation of such organelle-specific mark-ers will likely provide us with a deeper understanding ofthe structure, assembly, dynamic activity, and functionof this unusual subcellular structure that is one of thehallmarks of the Kinetoplastida.

4.1. Secretory pathway

Neither the secretory nor endocytic pathways arenearly as well understood in trypanosomatid protozoaas they are in mammalian cells or yeast, but the currentlevel of knowledge suggests that there are many broadparallels. In particular, there is an extensive endoplas-mic reticulum (ER) that is widely dispersed throughoutthe cytoplasm [29,53], there is a Golgi apparatus con-sisting of 4–6 flattened stacks, there is a ‘budding zone’of vesicles between the ER and the cis-face of theGolgi, there are flattened cisternae, tubulovesicular ele-ments and coated vesicles adjacent to the Golgi thatprobably constitute the trans-Golgi network, and thereare various larger vesicles (�100 nm) between thetrans-Golgi and the flagellar pocket that are thought tobe transport vesicles [54] (Fig. 5). Trafficking of theabundant variant surface glycoproteins (VSGs) hasbeen studied in bloodstream African trypanosomes [29],where these glycosylphosphatidylinositol (GPI) an-chored proteins form a dense coat that covers thesurface of the parasite and protects it from the hostimmune system [55]. Immunogold labeling of thincryosections demonstrated the presence of VSG in theER, all of the Golgi cisternae, the trans-Golgi network,and the transport vesicles, suggesting that newly synthe-sized VSG traffics sequentially through these organelleson its way to the flagellar pocket and cell surface, whichare also heavily labeled.

Studies on the extracellular enzyme secreted acidphosphatase (sAP) in promastigotes and intracellularamastigotes of L. dono�ani, the major secreted protein


Fig. 5. Ultrastructure of L. mexicana promastigotes prepared by high-pressure freeze substitution and Epon embedding showing a variety oforganelles involved in secretion or endocytosis (reproduced from [54] by copyright permission of The Company of Biologists Ltd.). (A) Anteriorend of the cell showing the Golgi (g), the ER (er), the electron dense budding zone (bz) with vesicles on the cis-side of the Golgi and withtranslucent vesicles (v) on the trans-side of the Golgi. (B) ER-derived vesicles in the budding zone (bz) appear to form new Golgi cisterna. (C)Vesicles filled with particles (asterisks) near the trans-side of the Golgi. Four coated vesicle-like structures (arrowheads) budding from the mosttrans-side cisterna of the Golgi. (D) Large spherical translucent vesicle (v) adjacent to the flagellar pocket. Below is an enlarged detail of the vesiclemembrane (large arrowhead) displaying a regularly arranged coat-like structure (small arrowheads). Tubules are often connected to largetranslucent vesicles (v, arrow and also arrows in E and F). (E and F) Clusters of regularly arranged tubules (t) located close to the Golgi (g) andthe flagellar pocket (fp) showing tubules in longitudinal section (E) and cross section (F). (G) Coated pit-like invaginations of the flagellar pocketmembrane. bz, budding zone; er, endoplasmic reticulum; fl, flagellum; fp, flagellar pocket; g, Golgi; k, kinetoplast; n, nucleus; t, clustered tubules;v, translucent vesicles. Bars are 0.5 �m (A–F) and 100 nm (inset D and G).

of this parasite, have underscored the role of the Golgiin modification of secreted proteins. Immunofluores-cence localization revealed that sAP was present dif-

fusely in the cytoplasm and in a more concentratedform in the flagellar pocket, and the enzyme was ulti-mately secreted into the medium [56], suggesting that it


is transported via a constitutive secretory pathway tothe lumen of the flagellar pocket and thence to theextracellular space. The extracellular enzyme waspresent as two heterodisperse bands of �110 and 130kDa on SDS-polyacrylamide gels but was synthesizedas two intracellular precursors of discrete sizes, 92.5and 107 kDa [57]. Addition of monensin, a classicalinhibitor of Golgi function, caused morphologicalchanges to the Golgi apparatus, as earlier shown in T.brucei [29], and suppressed the heterodisperse mobilityof the sAP bands [58], apparently by inhibiting a Golgi-specific carbohydrate modification. This and other [52]work has demonstrated that complex phosphoglycansare added to sAP in the Golgi complex and is responsi-ble for the diffuse mobility on SDS-polyacrylamidegels, and that similar modifications occur on othersecreted macromolecules including the abundant glycol-ipid lipophosphoglycan (LPG) and on a complex ofproteoglycans designated proteophosphoglycans (PPG)[59]. Furthermore, a GDP-mannose transporter re-quired for addition of mannose to complex carbohy-drates such as those on LPG is localized to the Golgi,where it imports from the cytoplasm into the Golgilumen the nucleotide sugar precursor used in synthesisof the oligosaccharide chains [60]. Hence as in mam-malian cells, the Golgi is responsible for complex car-bohydrate modifications during biosynthesis of secretedand membrane bound proteins and other macro-molecules.

Little is known about the precise nature of vesiclesthat mediate transport of secreted and membraneproteins between the ER, the Golgi, and the flagellarpocket. In general few markers are available for suchvesicles, and in vitro fusion systems have not beendeveloped to allow dissection of components requiredfor vesicle docking and fusion. However, genes encod-ing rab protein homologues, small G proteins involvedin regulating vesicle fusion in various steps of theexocytic and endocytic pathways of mammalian cells[61], have been cloned from T. brucei [62] supportingthe likely conservation of vesicle fusion mechanismsbetween these protozoa and higher eukaryotes.

4.2. Endocytic pathway

All endocytic events, either receptor-mediated orpinocytotic, appear to initiate at the membrane of theflagellar pocket. Invaginations resembling coated pits ofhigher eukaryotes can bee seen pinching off from theflagellar pocket surface [51,54] (Fig. 5G) and are likelyto be the precursors for endocytic vesicles. Followingexposure of bloodstream trypanosomes to antiseraagainst VSG, to HRP, or to labeled Tf, BSA, orferritin, a variety of internal vesicles and tubulovesicu-lar networks were labeled with the internalized compo-nents [51,63–68]. These membrane bounded bodies

ranged in diameter from 20 to 200 nm, were locatedbetween the flagellar pocket and the nucleus, and arebelieved to represent the early and late endocytic com-partments. In addition, internalized labeled materialalso appears in electron-dense vesicles thought to belysosomes. Coated vesicles from T. brucei, at least someof which are likely to be endocytic, have been isolatedand partially characterized. These vesicles contain aminor protein component with the same mobility onSDS-polyacrylamide gels as bovine brain clathrin, butthis protein does not cross-react with antibodies di-rected against mammalian clathrin, and its identity isuncertain [69]. An apparent integral membrane proteinof 77 kDa has been isolated from these vesicles, andantisera against the gel-purified protein labels vesicles inthin sections of trypanosomes, suggesting that thispolypeptide is a marker for endocytic vesicles [70].

A number of studies have also been performed onendocytosis in promastigotes and amastigotes of severalLeishmania species. Overath and colleagues [54] haveapplied an improved sample preparation method ofhigh pressure freezing and freeze-substitution to visual-ize vesicles and tubules in the anterior region of L.mexicana promastigotes. In addition to observing im-proved images of organelles involved in secretion, suchas the ER, the Golgi, intermediary transit vesicles, andlarge translucent vesicles close to the flagellar pocket(Fig. 5), they were able to identify various membranousstructures that labeled with the fluid phase markerHRP, with biotinylated surface markers, or with antis-era against several membrane proteins or oligosaccha-rides and thus represent the endocytic pathway. Inaddition to a rich array of vesicles and tubules similarto those observed earlier, a thicker tubule �100–200nm in diameter was observed that extended the lengthof the cell, was itself filled with smaller vesicles, and waslabeled with internalized components. This multivesicu-lar tubule (MVT) labeled with antisera directed againstboth a transmembrane form and a GPI-anchored formof acid phosphatase and with the GPI-anchored surfaceprotease GP63, but it did not label with an antibodydirected against the protein component of sAP or thesecreted PPG. These latter results suggest that MVT isnot part of the secretory pathway, as had been pro-posed originally (at the time of the initial discovery ofthe MVT) on the basis of light microscopic data relat-ing to overexpression of dolichol–phosphate–mannosesynthase, a normally ER-resident enzyme involved inGPI biosynthesis [71]. Rather the MVT appears to bepart of the endocytic machinery that internalizes sur-face proteins that are expressed at relatively high levels,including the overexpressed proteins used in these stud-ies [54,71,72]. Presumably, proteins transit from theflagellar pocket to the MVT via other vesicles andtubules observed by Overath and colleagues. Similarconclusions have been reached by Dwyer and col-


leagues [72] by studying high level expression in L.dono�ani promastigotes of GFP fusion proteins con-taining either the transmembrane domain of 3� nucleoti-dase/nuclease or the GPI addition signal of GP63.These tagged fusion proteins trafficked to the cell sur-face but were also found in an MVT compartmentrunning the length of the cell. Fluid phase and particu-late markers such as dextrans and positively chargednanogold particles, used to track endocytosis in highereukaryotes, were also internalized into the same MVTstrongly suggesting an endocytic nature for thiscompartment.

As in higher cells, lysosome-like organelles appear tobe the end stage of the endocytic pathway for manyinternalized components. In the L. mexicana family,two lysosomal enzymes, cysteine proteinase and arylsul-fatase [73], are localized to large electron dense or-ganelles originally designated ‘megasomes’ [74] that areconsidered to be the unusual lysosomes of these para-sites. Furthermore, biotinylated markers such as dex-tran or �-glucorinadase were internalized by L.mexicana infected macrophages and subsequently en-tered the parasitophorous vacuole and the flagellarpocket and were thence targeted within the parasite tothe megasomes [75].

4.3. LDL receptors and the CRAM protein

LDL is required by African trypanosomes for robustgrowth and apparently is the major source of choles-terol for these parasites [76]. Early biochemical studies[51] demonstrated that LDL uptake is saturable, can becompeted by unlabeled LDL, is temperature- andCa+2-dependent, and occurs at a very rapid rate com-pared with fluid-phase uptake. Furthermore, gold la-beled LDL adhered to the flagellar pocket membraneand was also present in intracellular vacuoles, suggest-ing that a specific LDL receptor was located in theflagellar pocket and mediated internalization of thislipoprotein. A T. brucei protein of 86 kDa, apparentlya fragment of a larger 145 kDa protein [77], has beenpurified by LDL affinity chromatography and proposedto be the parasite LDL receptor [76], although it hasalso been suggested [78] that this polypeptide may be acontaminant that co-purifies with the true receptor. Apolyclonal antiserum against the purified fractionstained the flagellar pocket, and much of the stainappeared to be in the lumen of the pocket.

In separate work [79], the gene for the cysteine-rich,acidic, integral membrane protein (CRAM) was cloned

Fig. 6. Localization of CRAM protein in the flagellar pocket (fp) membrane of a procyclic (insect form) T. brucei cell (reproduced from [79] bycopyright permission of the American Society for Microbiology). The electron dense spots adjacent to the membrane are 15 nm gold particles thatwere used for indirect immunolabeling of CRAM protein. The arrow marks the flagellum.


and shown to encode a protein with multiple 12 aminoacid repeats that have substantial sequence similarity tothe repeat units in the NH2-terminal region of the humanLDL receptor. Furthermore, CRAM is located on theflagellar pocket membrane (Fig. 6), as determined byimmunoelectron microscopy, and the repeat units appearto be on the lumenal side of this membrane. Hydropathyanalysis of the sequence revealed the presence of a singleputative transmembrane segment downstream of therepeat units followed by a hydrophilic COOH-terminaldomain of 41 amino acids. CRAM RNA is expressed atabout a 5-fold higher level in procyclic trypanosomescompared with bloodstream forms, and the CRAMprotein is also expressed more abundantly in procyclicparasites. On the basis of the similarity to mammalianLDL receptors, it has been suggested [80] that CRAMmight be a parasite receptor for a lipoprotein, possiblyhigh density lipoprotein (HDL).

The higher level of expression of CRAM in procyclicparasites compared with bloodstream forms has raisedan interesting question. Coated pits and vesicles typicallyassociated with receptor-mediated endocytosis have onlybeen observed in bloodstream and not procyclic try-panosomes [64], and it has been suggested that receptor-mediated endocytosis may be of minimal importance inthe insect stage of the life cycle, where CRAM isexpressed at the higher level. However, studies on pro-cyclic trypanosomes have demonstrated that anti-CRAM IgG can bind to and be endocytosed by theCRAM protein during this life cycle stage [80]. Thesestudies reveal that endocytosis followed by degradationof the ligand does occur in procyclics, possibly viavesicles of distinct morphology from those seen in themammalian stage of the life cycle.

An important study on CRAM by Lee and colleagues[81] has contributed to our understanding of targetingand retention of flagellar pocket membrane-specificproteins. These authors observed that a deletion of eithereight or 19 amino acids from the COOH-terminus ofCRAM caused the protein to be retained in the ER,where it largely localized with the ER marker Bip.Unexpectedly, deletions of either 29 or 40 amino acidsfrom the COOH-terminus partially restored traffickingpast the ER but allowed the surface protein to reach theflagellum and pellicular plasma membrane, as well as theflagellar pocket membrane. The authors suggest that asignal for transit through the ER is present in the firsteight amino acids and that a signal for retention in theflagellar pocket is encompassed by the first 29 aminoacids. Although the precise explanation for the behaviorof the deletion mutants is not clear, removal of bothsignals apparently allows some of the protein to trafficto the cell surface but not to be retained at the flagellarpocket. Consistent with the presence of targeting infor-mation within the 41 amino acid COOH-terminal do-main, a fusion between the TrpE protein and this

cytoplasmic domain of CRAM traffics to the flagellarpocket. Intriguing problems that remain to be solved areto define the precise amino acids that constitute theflagellar pocket retention signal and to identify theproteins that presumably interact with this signal toprevent further migration of CRAM over the surface ofthe parasite.

4.4. The T. brucei transferrin receptor

Early biochemical studies demonstrated that Tf istaken up by a mechanism resembling receptor-mediatedendocytosis and that Tf-gold was localized to the flagellarpocket with staining largely in the lumen of this organelle[51]. Subsequent work by Schell et al. [82] succeeded inpurifying a 42 kDa Tf-binding protein (TFBP) by Tf-affinity chromatography of solubilized membranes fromT. brucei bloodstream forms that was ultimately iden-tified as the product of the expression site-associated gene7 (ESAG 7) [83] that is located in telomeric expressionsites upstream from expressed copies of the VSG genes[7]. The open reading frame of ESAG 7 is closely relatedto that of ESAG 6, and subsequent studies [84] confirmedthat the functional TFBP is a heterodimer of ESAG 6and ESAG 7. Thus expression of both gene products wasrequired for Tf binding when expression studies wereperformed in Xenopus oocytes [85], insect cells [86], orprocyclic trypanosomes [87]. ESAG 7 is not an integralmembrane protein, and ESAG 6 is modified by a GPImoiety. The receptor oligomer is apparently held into themembrane by the ESAG 6 GPI anchor. Furthermore,immunoelectron microscopy studies [85,87,88] with anti-sera directed against ESAG 6 or ESAG 7 revealedstaining largely in the lumen of the flagellar pocket butalso on the flagellar pocket membrane. Further supportfor TFBP as a bonafide Tf receptor comes from theobservation that TFBP complexed to Tf is routed to thelysosomes where the Tf is degraded but the TFBP isrecycled [84].

The striking difference between the structure of theoligomeric GPI-linked T. brucei TFBP and the mam-malian Tf receptor, which is a transmembrane protein,suggests that these two types of receptor function infundamentally different ways. First, classical receptorsinvolved in endocytic events contain a cytoplasmic do-main required for ligand-dependent internalization [89],whereas neither ESAG 6 nor ESAG 7 possesses such adomain. It is possible that the TFBP interacts withanother transmembrane protein to transmit the ligand-induced signal for internalization. Furthermore, theconsistent observation that TFBP is largely present in thelumen rather than in the membrane of the flagellarpocket is puzzling and raises the question of how sucha protein might function. Possibly, TFBP is released intothe lumen to bind Tf and then either re-enters themembrane or transfers its ligand to membrane bound


Fig. 7. Fibrous material in the lumen of the flagellar pocket detected in a deep-etch, freeze fracture image of the flagellar pocket of an L. mexicanapromastigote (reproduced from [52] by copyright permission of The Rockefeller University Press). The filaments (arrowheads) are composed ofparticles with a similar size and periodicity to those of purified sAP and thus may represent in situ polymers of this protein. The arrow pointstoward the opening of the flagellar pocket, and fl marks the flagellum.

receptors. Thus fundamentally important questions thatremain to be answered are — how is the TFBP inter-nalized, how is it retained in the flagellar pocket, andwhat is the relationship between apparently soluble andmembrane bound TFBP? The mechanism of Tf uptakeis another example of the intriguing differences betweenthese single cell parasites and their more complex mam-malian hosts.

4.5. Possible hemoglobin receptor

Leishmania parasites require hemin for growth, butthey are able to grow on blood agar medium withoutaddition of hemin, suggesting that they can acquire thisnutrient from hemoglobin. Biochemical and ultrastruc-tural studies [90] have demonstrated that promastigotesof L. dono�ani bind 125I-hemoglobin with high affinity,that this binding can be specifically competed withunlabeled hemoglobin, and that the hemoglobin bindsinitially to the flagellar pocket membrane and is subse-quently internalized in vesicles and degraded. Affinitychromatography led to the isolation of a 46 kDaprotein derived from a membrane fraction that boundto hemoglobin agarose but whose binding could bespecifically competed with unlabeled hemoglobin.Hence, this 46 kDa protein may be a hemoglobinreceptor that is localized to the flagellar pocketmembrane.

4.6. Proteins secreted into the lumen of the flagellarpocket

Since proteins destined for secretion reach the cellsurface at the flagellar pocket, the lumen of the pocketfunctions as an intermediate transit zone between thecytoplasm and the extracellular space, and work fromvarious groups has demonstrated that several secretedproteins accumulate in the pocket before their release.Immunofluorescence studies on the abundantly releasedsAP of L. dono�ani promastigotes revealed that thisenzyme accumulates to a high concentration in theflagellar pocket and that release from the pocket wasdependent upon energy and may be coupled to flagellarbeating [56]. A conserved sAP is secreted by manyspecies of Leishmania [91] and by both promastigotesand amastigotes [92]. Overath and colleagues [52] havealso studied sAP extensively in L. mexicana promastig-otes. In this species, sAP consists of a 100 kDa glyco-protein associated non-covalently with a high molecularweight proteoglycan, and assembles into striking longcurved filaments that can be imaged by electron mi-croscopy and are composed of bead-like subunits.These sAP filaments can be found abundantly in thelumen of the flagellar pocket (Fig. 7) and are secretedinto the extracellular medium. The authors suggest thatmonomers or oligomers are initially secreted into thepocket where they assemble into the filaments and


ultimately exit the pocket. One hypothesis emerging fromthese studies is that the flagellar pocket lumen is a sitefor assembly of polymeric macromolecular structures,much the way that collagen fibrils and other componentsof the extracellular matrix assemble in extracellularspaces in mammalian tissues. In contrast, the sAP of L.dono�ani promastigotes was non-polymeric and revealedglobular mono- or oligomers in negatively stained elec-tron micrographs [93]. Cloning of sAP genes from L.mexicana identified two genes, lmsap1 encoding themajor 100 kDa sAP and lmsap2 encoding the minor 200kDa sAP [94]. In both enzymes, Ser/Thr-rich domainswere the sites for extensive phosphoglycan modification.Similarly two tandemly repeated sAP genes, SAcP-1 andSAcP-2, were cloned and sequenced from L. dono�aniand shown to encode isoforms with regions of highsequence identity, including Ser/Thr-rich domains be-lieved to be the sites of phosphoglycan addition [95].

A second type of filamentous structure originallycalled the ‘network’ is clearly distinct from sAP, and isalso secreted into the flagellar pocket lumen [52]. Net-work material emerging from the mouth of the flagellarpocket appears as a meshwork in the center of clustersof promastigotes that adhere to each other in culture andin the insect and may be responsible for formation ofthese large cellular aggregates. These network fibers stainwith a monoclonal antibody directed against a carbohy-drate epitope that is also present in sAP, but they do notstain with a monoclonal antibody directed against thepolypeptide component of sAP. More recently, thenetwork filaments of L. major have been studied at themolecular level. Purification of material secreted into themedium followed by structural analysis revealed a veryunusual proteophosphoglycan (PPG) that ran as a dif-fuse, high molecular weight species on SDS-polyacry-lamide gels, contained large amounts of mannose,galactose, arabinose, and phosphate, and whose proteincomponent consisted of 87 mol% of glycosylated phos-phoserine, serine, alanine, and proline [96]. The glycansisolated from PPG were also components of the majorsurface glycolipid LPG but were organized differently,and they were also present in sAP, indicating that bothglycoproteins undergo the novel Golgi-mediated glycosy-lation. In vitro this purified material formed long (up to6 �m), cable-like, unbranched filaments that were indis-tinguishable from the ‘network’ filaments earlier iden-tified. In L. major there are multiple genes encodingrelated PPGs, and one of these, ppg1, was the first to becloned and sequenced [97]. The predicted protein productof this gene (�2300 amino acids) contains NH2-terminaland COOH-terminal domains with conventional aminoacid sequences separated by a region containing �100repetitive peptides consisting exclusively of alanine, ser-ine, and proline that are the sites of phosphoglycanattachment. Remarkably, the ppg1 gene product is amembrane bound protein (mPPG) that is modified by a

GPI anchor and is distributed over the pellicular plasmamembrane, indicating that secreted PPG must be en-coded by other members of this gene family. Subsequentwork [59] has led to the cloning of candidate sequencesfor the secreted filamentous PPG, now designated fPPG,and a nonfilamentous PPG secreted by amastigotes,designated aPPG.

Elegant in vivo studies [98] have established a likelyrole for secreted fPPG in the parasite-vector interaction.Both L. mexicana infecting Lutzomyia longipalpis and L.major infecting Phlebotomus papatasi form parasite ag-gregates in the thoracic midgut and stomodeal valve thatare held in place by a gel-like plug. Immunoelectronmicroscopy revealed that this plug consists of filamen-tous networks that entangle and immobilize the pro-mastigotes and that stain with antisera that are specificfor PPG. This matrix is proposed to both retain theparasites within the insect gut following digestion of theblood meal and to block access between the foregut andthe midgut during a subsequent blood meal. The conse-quent difficulties the sandfly experiences engorging theblood lead to multiple probing of the host accompaniedby increased disgorging of infectious metacyclic formsthat are largely free of the network and located anteriorto it. Hence, the promastigotes secrete filamentous mate-rial from the flagellar pocket that profoundly influencesthe vector-parasite interaction to promote parasite trans-mission.

Finally, a recent study [99] has demonstrated that bothGPI-linked proteins that are otherwise anchored into theplasma membrane and phospholipase C-cleaved GPI-linked proteins that have had their lipid anchors removedare present in the lumen of the flagellar pocket. Antiseraspecific for the cleaved GPI anchor (earlier called cross-reacting determinant) react with flagellar pocket compo-nents both before in vitro treatment with phospholipaseC (representing species whose anchors were earliercleaved in vivo) and after in vitro treatment with thisenzyme (representing intact GPI-containing species thatwere cleaved in situ within thin sections). It is not clearhow GPI-containing proteins, including the Tf receptorand VSG, are released from the membrane into the lumenof the pocket or what function they might serve there,but they may exist as micellar structures explaining theirsolubility in an aqueous environment.

4.7. Trypanosome T lymphocyte triggering factor

In studies on the interaction of T. brucei parasites withtheir mammalian hosts, a parasite gene was isolated [100]that encodes a protein designated T lymphocyte trigger-ing factor (TLTF) that induces CD8+ T cells to secreteinterferon-�. This protein does not contain an apparentsignal sequence and hence does not appear to follow theclassical biosynthetic pathway for a secreted, vesicular,or integral membrane protein. Fluorescence microscopy


localization using GFP fusions to TLTF revealed thatthis protein is targeted to the region of the flagellarpocket at the base of the flagellum. More detailedlocalization by immunoelectron microscopy showed thatthe fusion protein was enclosed in electron dense bodiesthat subtended the flagellar pocket [101]. Thus althoughTLTF appears not to be a membrane protein, it is presentinside ovoid shaped bodies that may be vesicular and thatare closely associated with the flagellar pocket mem-brane. Furthermore, these electron dense bodies clusterat the anterior side of the flagellar pocket, suggesting thatTLTF interacts asymmetrically with the flagellar pocketand also implying that the flagellar pocket membrane islikely to be non-uniform in structure. The biologicalsignificance of this subcellular location is not clear, norhas the function of TLTF been determined.

Donelson and colleagues have investigated the regionof TLTF responsible for the flagellar pocket-associatedtargeting by preparing chimeras between segments ofTLTF and GFP, and they have identified a large segmentspanning amino acids 114–257 of this 453 amino acidprotein that is responsible for the described localization.Random mutagenesis of this targeting domain revealedmany mutations that did not affect targeting and othermutations scattered throughout the domain that didaffect localization. Thus, the 144 amino acid domainappears to represent a three dimensional (3-D) structuraltargeting signal and clearly does not encompass a shortcontiguous motif of the type responsible for localizationof many organelle-specific proteins. This region of TLTFis predicted to have a high �-helical content and a highprobability of forming a coiled-coil, suggesting thatsecondary structure of this type could be involved in therouting of this polypeptide. One very intriguing result isthat a subset of the targeting mutations cause mislocal-ization of the mutant protein not to the cytoplasm butto the flagellum, with especially strong staining at theanterior tip of this organelle. The authors suggest thepossibility that TLTF normally interacts with subpellic-ular microtubules during its trafficking to the flagellarpocket, and that some disruption of a retention signal inthe aforementioned mutants allows them to continue totraffic along the adjacent flagellar microtubules, resultingin their movement along the body of the flagellum to itsanterior tip.

More recent investigations [102] have shown thatTLTF is a cytoskeleton-associated protein that fraction-ates quantitatively with the insoluble cytoskeletal pelletupon lysis of cells with nonionic detergent. Furthermore,the protein appears to be associated with the flagellarfraction of the cytoskeleton, as solubilization of thepellicular microtubules with Ca+2 does not extractTLTF but leaves it behind in a pellet containing theflagellar axoneme, paraflagellar rod, and other associatedfibrous structures such as the flagellar attachment zoneand a quartet of specialized microtubules that line the

anterior face of the flagellar pocket. Hence, TLTFappears to target to the anterior cytosolic face of theflagellar pocket by specific association with a subset ofcytoskeletal components. The results with TLTF high-light the complexity of subcellular targeting events thatexist in trypanosomatids and broaden the scope ofcurrent investigations to include a protein that probablydoes not associate directly with a membrane.

5. Overview and future directions

The last few years have seen a mini-explosion in theidentification of proteins that target to the flagellum andflagellar pocket in trypanosomatid protozoa. We nowhave available several reasonably well-characterizedproteins that are routed to various components of eachorganelle, and in some cases, we have information on thesequence within the protein that is responsible for thesubcellular localization. There are a number of issuesraised by these studies that are now ripe to be addressed.

In the first instance, it will be important to identify thecellular machinery that interacts with each protein andthat delivers it and/or retains it at the correct intracellularsite. However, one potential difficulty of genetic strate-gies will be to design screens or selections that will detectmutations that alter delivery of flagellar or flagellarpocket proteins. Targeting processes that are operativein other organisms, such as IFT, can be investigated intrypanosomatids to determine whether they are respon-sible for routing of proteins to the flagellum or flagellarpocket in these protozoa as well. It will also be interestingto determine how widely targeting signals can be recog-nized among different trypanosomatids or even in otherorganisms. The unique structure of these protozoa sug-gests that there may be some highly specialized systemsthat have been elaborated by these parasites to constructand maintain their own distinctive organelles. However,it is also likely that some of the routing pathways broughtto light in these ancient eukaryotes will prove to berelevant to distantly related organisms, as was brilliantlyproven with the initial discovery of GPI anchors intrypanosomes [55] and their subsequent identification inmammalian cells.

Another area of active investigation is to furtherelucidate the biological functions of flagellar and flagellarpocket proteins. In most cases, we have hints or partialknowledge about possible activities but not a clearpicture of the specific biochemical or physiological func-tions or the reasons for the sequestration of the proteinswithin each organelle. Thus, the ISO1 protein canoperate as a glucose transporter, but we do not yet knowwhy it is targeted to the flagellar membrane, whileanother very similar permease traffics to the pellicularplasma membrane. While the flagellar receptor-adenylatecyclases and the flagellar calcium-binding proteins are


almost certainly involved in signal transduction, we donot know what initial signals they respond to nor theparticular pathways in which they are involved. It hasnow been determined that the flagellar rod proteins areoperative in some significant way in flagellar motility, buthow they contribute to this activity is not at all clear.While CRAM is most likely involved in receptor-medi-ated endocytosis, the ligand recognized by this putativereceptor has yet to be definitively identified. The role ofTLTF in the biology of the trypanosome is still a mystery,as it is unlikely to serve the parasite well by simplyactivating T cells to secrete �-interferon. Nonetheless, itis likely that further research on these well-defined‘markers’ of flagella and flagellar pockets will give usconsiderable additional insight into the specialized func-tions of each organelle. Thus, we have been alerted to theprobable function of flagella in sensing the extracellularenvironment in addition to their ‘conventional’ functionin parasite motility. We are likely to gain a deeperappreciation for the multiple activities of these complexorganelles that must often serve biological functions inthese ‘simple’ unicellular eukaryotes that can be achievedby multiple differentiated cells in metazoan organisms.

Acknowledgements

This work was supported by grant number AI25920from the National Institutes of Health. S.M.L. is aBurroughs Wellcome Molecular Parasitology Scholar,and M.I. is a postdoctoral fellow of the American HeartAssociation, Northwest Chapter.

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