1 Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, WI 53706, USA
2 Department of Biomolecular Chemistry, University of Wisconsin-Madison Medical School, Madison, WI 53706, USA
* Author for correspondence (e-mail: jdbangs{at}wisc.edu)
Accepted 31 August 2005
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Summary |
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Key words: Trypanosome, Glycosylphosphatidylinositol, Flagellar pocket, Transferrin receptor, Protein sorting
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Introduction |
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Not surprisingly, given its abundance and its importance to pathogenesis, VSG is the most studied protein in African trypanosomes. Newly synthesized VSG is rapidly modified in the endoplasmic reticulum by N-glycosylation and by attachment of the C-terminal GPI-anchor (Bangs et al., 1985; Ferguson et al., 1986
). Homodimerization occurs during this early stage of transport (McDowell et al., 1998
; Triggs and Bangs, 2003
). During subsequent transport from the ER and through the Golgi both GPI and N-glycans are modified, and thereafter VSG is incorporated via the flagellar pocket into the surface coat with an overall transport rate of t1/2
15 minutes (Bangs et al., 1986
; Bangs et al., 1988
; Duszenko et al., 1988
; Mayor et al., 1992
). VSG recycles constantly between the plasma membrane and internal endosomal compartments (Overath et al., 1997
), yet is remarkably stable in bloodstream trypanosomes (t1/2 >30 hours) (Bulow et al., 1989
; Seyfang et al., 1990
). However, the rate of membrane uptake within the flagellar pocket is exceedingly rapid (an entire surface coat equivalent in <12 minutes) (Engstler et al., 2004
; Grünfelder et al., 2003
), thus sorting back to the surface must be efficient to avoid degradation in the lysosome. Recent studies have defined the intracellular pathway of VSG sorting (Engstler et al., 2004
; Grünfelder et al., 2003
). Uptake is via clathrin-coated vesicles (Class I) followed by delivery to a RAB5+ early endosomal compartment. After maturation into a RAB11+ recycling endosome, lysosomal cargo is distilled into small clathrin-coated vesicles (Class II), leaving behind VSG+ exocytic carriers that fuse with the flagellar pocket. Within this pathway there is a steep gradient of lateral VSG density moving from internal compartments to the plasma membrane [ER (1x), Golgi (3x), flagellar pocket/plasma membrane (50x), endosomes (10x)] (Grünfelder et al., 2002
). Unlike VSG, nothing is known about the transport and turnover of endogenous procyclins in insect stage trypanosomes. Nevertheless it is assumed that GPI anchors strongly influence membrane protein trafficking in both life cycle stages.
The only other GPI-anchored protein to be studied in trypanosomes is the bloodstream stage-specific transferrin receptor (TfR). TfR is a heterodimer of the ESAG6 and ESAG7 gene products (Ligtenberg et al., 1994; Salmon et al., 1994
). These subunits have high sequence similarity with each other, as well as overall structural similarity to VSGs (Carrington and Boothroyd, 1996
), but only ESAG6 is GPI-anchored. The steady state location of this single GPI heterodimer is in the flagellar pocket; it is not normally detectable on the external plasma membrane. However, modest overexpression induced by transferrin starvation leads to `spill-over' onto the cell surface (Mussman et al., 2004
; Mussman et al., 2003
), TfR is continuously endocytosed bearing serum transferrin, which is delivered to the lysosome, and the receptor is recycled to the flagellar pocket. The half-life of TfR has been variously estimated to be 0.7-7.0 hours (Biebinger et al., 2003
; Kabiri and Steverding, 2000
; Mussman et al., 2004
) and turnover apparently occurs in the lysosome (Steverding et al., 1995
).
We have previously studied the role of GPI anchors in trypanosomes by expression of modified GPI-minus VSG reporters (Bangs et al., 1997; McDowell et al., 1998
; Triggs and Bangs, 2003
). In procyclic trypanosomes GPI-minus VSG is delayed in ER exit, but is ultimately secreted quantitatively. In bloodstream trypanosomes these reporters are also apparently delayed in ER exit, but once exit is achieved they are delivered to and degraded in the lysosome (t1/2
1 hour depending on the VSG). Similar results were obtained with an analogous GPI-minus version of TfR (Biebinger et al., 2003
) and with VSGs (Böhme and Cross, 2001
). Collectively these findings suggest that GPI anchors function in the early secretory pathway, and that in bloodstream parasites GPIs also play a critical role in post-Golgi targeting to the cell surface.
Our findings, in conjunction with the known behavior of native VSG and TfR present an intriguing correlation between GPI valence and the ultimate fate of proteins within the secretory pathway of bloodstream trypanosomes: native VSG (GPI2) has a steady state cell surface localization and is exceptionally stable; TfR (GPI1) is located in the flagellar pocket and is turned over more rapidly; and GPI-minus VSG (GPI0) is delivered to the lysosome and degraded most rapidly. We now present data in bloodstream trypanosomes with a series of proteins of the native secretory/endocytic pathways engineered to have a valence of GPI1 and GP0, and also with native TfR. These reporters conform generally to the `valence correlation', but surprisingly we find that GPI1 membrane proteins can also be released from viable bloodstream trypanosomes with intact GPI anchors. A simple model is proposed for parsing these reporters between the alternate fates of release versus lysosomal degradation, and the implications of our novel findings for trafficking of native GPI-anchored proteins in trypanosomes are discussed.
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Materials and Methods |
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Immunoprotocols, antibodies and electrophoresis
Immunoprecipitation and electrophoresis have been described previously (Bangs et al., 1997; McDowell et al., 1998
; Triggs and Bangs, 2003
). The only modification was that for immunoprecipitation of EP procyclin reporters cells were solubilized in 1 x RIPA salts (50 mM TrisHCl, pH 8.0, 100 mM NaCl) containing 0.5% SDS, denatured (5 minutes, 95°C), and then adjusted to 1 x RIPA salts with 1% NP40, 0.5% deoxycholate, 0.1% SDS. All cell lysates and media fractions were supplemented with protease inhibitors (final concentrations 2 µg/ml each of leupeptin, antipain, chymostatin and pepstatin, and 0.1 mM tosyllysine chloromethyl ketone). Rabbit and mouse anti-BiP, rabbit anti-VSG221 and monoclonal anti-p67 are described in (Alexander et al., 2002
; Bangs et al., 1996
; McDowell et al., 1998
). Monoclonal 247 anti-EP procyclin was purchased from Cederlane Laboratories Ltd. (Hornby, Ontario). Rabbit anti-HA and monoclonal 12CA5 anti-HA were purchased from Zymed (San Francisco, CA) and BabCO (Richmond, CA), respectively. Rabbit anti-EP procyclin was prepared by immunization with a synthetic (EP)9 peptide coupled to keyhole limpet hemocyanin (anti-EP9). Monoclonal anti-Leishmania paraflagellar rod and rabbit anti-transferrin receptor were kindly supplied by Diane McMahon-Pratt (Yale University) and Piet Borst (Netherlands Cancer Institute, Amsterdam), respectively. Biotinyl-tomato lectin was purchased from Vector Laboratories, Burlingame CA.
Gels were exposed on a Molecular Dynamics Typhoon system and phosphorimages were quantified in the native ImageQuant Software (Amersham Biosciences, Piscataway, NJ). For each lane, densities of identical regions of interest were measured and corrected by subtraction of the density for an equivalent unlabeled region.
Construction of reporter genes
All reporters are shown diagrammatically in Fig. 1 and were cloned into the constitutive expression vectors pXS5neo (Alexander et al., 2002) using flanking 5 ' HindIII and 3 ' EcoRI sites. Vector plasmids were linearized with XhoI and stably transformed bloodstream cell lines were generated as described in (Triggs and Bangs, 2003
).
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Immunoelectron microscopy
Fresh samples were embedded in 2% agarose in 0.1 M phosphate buffer (PB), pH 7.4 at 50°C. After cooling, samples were fixed (30 minutes) in 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M PB, sectioned into 100 µm slices, fixed again (2 hours), and then quenched with 0.1% sodium borohydride in 0.1 M PB (10 minutes). The slices were incubated in 0.1% Triton X-100 (30 minutes) and then blocked (1 hour, RT) with Aurion Goat Blocking Agent (Aurion, Wageningen, The Netherlands). After rinsing in incubation buffer [IB: PBS+0.1% BSA-c (Aurion)], slices were incubated in anti-EP (mAb247, 1:500 in IB, 2 hours at RT, then 16 hours at 4°C). Following extensive rinsing, slices were incubated with goat anti-mouse IgG F(ab')2 Ultra-Small gold conjugate (Aurion) (1:100 in IB, 2 hours at RT, then 16 hours at 4°C). Stained slices were rinsed sequentially in IB and PBS, and then postfixed in 2% glutaraldehyde in 0.1 M PB (30 minutes) and finally in PB. Postfixed slices were rinsed in Enhancing Conditioning Solution (ECS, Aurion) and then developed in Silver Enhancement Solution (Aurion) for 1.25 hours. Enhancement was terminated in 0.3 M sodium thiosulfate in ECS (5 minutes) followed by rinsing in ECS. Enhanced slices were further fixed with 0.5% osmium tetroxide in 0.1M PB (30 minutes) and then serially dehydrated in ethanol. Samples were transferred into propylene oxide and flat-embedded in Spurr's epoxy resin (Electron Microscopy Sciences, Hatfield, PA). Sections (60-90 nm) were cut on a Reichert-Jung Ultracut-E Ultramicrotome and contrasted with Reynolds' lead citrate and 8% uranyl acetate in 50% EtOH. Ultrathin sections were mounted and observed with a Philips CM120 electron microscope. All images were captured with a MegaView III (Soft Imaging System; Lakewood, CO) side-mounted digital camera.
Transferrin binding
BS221 cells (5x107), grown in HMI9 media supplemented with either 10% canine or fetal bovine serum for 48 hours, were washed with HEPES buffered-saline (HBS) and resuspended in 0.8 ml HMI9 media lacking serum but supplemented with 10 mg/ml bovine serum albumin to reduce nonspecific binding. Tf-gold (0.2 ml bovine holotransferrin conjugated to 5 nm gold suspended in PBS to an OD520 of 5.8, Aurion) was added and the cells were incubated at 8°C for 1 hour followed by washing in HBS to remove unbound Tf-gold. Cells were then fixed in 2.5% glutaraldehyde, 2.0% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for
20 hours at 4°C. The cells were post-fixed in 1% osmium tetroxide in the same buffer for 1 hour at RT. Samples were dehydrated, embedded, sectioned, and observed as described above.
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Results |
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Expression of procyclin reporters in bloodstream trypanosomes
Expression of the procyclin reporters in stable bloodstream cell lines was investigated by pulse radiolabeling in the presence and absence of tunicamycin, an inhibitor of N-glycan synthesis (Fig. 2A, top). No procyclin polypeptides were detected under either condition in cell or media fractions from untransformed control cells (lanes 1 and 2, 7 and 8). Furthermore, in all cell lines tested endogenous VSG 221 behaved as expected, a single species of 55 kDa that was reduced in size by tunicamycin treatment consistent with the loss of 2 N-glycans (lanes 1-6, bottom). No VSG was detected in the media fractions (Fig. 2A bottom, lanes 7-12).
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In untreated cells the EPMH reporter was detected as a sharp 40 kDa precursor band and a mature
55 kDa smear (lane 3, P and M). The precursor/product relationship of these glycoforms is confirmed in the following experiments. The precursor size was reduced
2 kDa by tunicamycin treatment consistent with utilization of the single N-glycosylation site (compare lanes 3 and 4). Tunicamycin treatment partially reduced the size of the mature form indicating that the N-glycan is modified, but that additional post-translational processing must also be occurring. This second mode of processing, the nature of which is unclear, is likely related to the GPI anchor, as a similar phenomenon was not observed with the otherwise identical GPI0 EPMH
gpi reporter (see below). Unexpectedly, the mature glycoform was also detected in the accompanying media fractions (lanes 9 and 10) suggesting that the reporter is exported from cells. Inhibition of N-glycosylation had no effect on release of mature EPMH (lane 10).
The EPMHgpi reporter was detected predominantly as an
28 kDa glycoform with trace amounts of higher molecular mass glycoforms (lane 5), and both forms were quantitatively reduced to a discrete
26 kDa band by tunicamycin treatment (lane 6), again consistent with utilization of the single N-glycosylation site followed by minor amounts of N-glycan processing. The little EPMH
gpi reporter detected in the media fractions was entirely of the larger processed form and this was quantitatively unglycosylated in the presence of tunicamycin (lanes 11 and 12).
To determine the nature of the EPMH N-glycan modifications radiolabeled reporter was first immunoprecipitated with anti-EP9, and then following solubilization reprecipitated with either anti-EP9 or with tomato lectin (Fig. 2B). Tomato lectin specifically binds the unusual giant poly-N-acetyllactosamine (pNAL) chains that are attached to N-glycans of many proteins in the endomembrane system of bloodstream trypanosomes (Atrih et al., 2005; Nolan et al., 1999
). Only the mature processed glycoform was detected by reprecipitation with tomato lectin (lane 3) indicating that the 40 kDa glycoform is an immature precursor to the larger fully modified glycoform. This experiment does not allow a definitive assignment of the site of addition of tomato-lectin-reactive pNAL, but this is likely to be the single N-glycan of EPMH given the precedent for this sort of modification of N-glycans in bloodstream trypanosomes, and the magnitude of the size shift seen in tunicamycin-treated cells, which is too large to be compatible with the loss of a more typical high mannose or complex oligosaccharide.
Finally, to establish that the EPMH reporter is actually GPI-anchored, cells were metabolically radiolabeled with [3H]Myristate and procyclin polypeptides were immunoprecipitated from cell and media fractions (Fig. 2C). Both glycoforms were detected in the cell fraction (lane 1), but surprisingly, radiolabeled mature glycoform was also detected in the media indicating that release is not dependent on GPI hydrolysis (lane 2) by endogenous GPI-phospholipase C (GPI-PLC). As expected [3H]Myristate-labeled VSG 221 was only detected in the cell-associated fraction (compare lanes 3 and 4). These results confirm that the procyclin GPI attachment sequence functions in bloodstream trypanosomes, and suggest that proteins with a GPI1 valence can be released from bloodstream trypanosomes with an intact GPI anchor. No labeling of the EPMHgpi reporter was detected (data not shown) indicating that myristate incorporation was GPI-dependent.
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Analyses of the EPMHgpi cell line indicates that this
28 kDa reporter also disappears from cells during the chase (Fig. 3B, lanes 1-5), but very little of the reporter is actually secreted into the media fraction (lanes 6 and 7, 17%). Treatment with FMK024 leads to greatly elevated recovery of cell-associated reporter (8% versus 52%), much of which is detected as a higher molecular mass smear (lanes 8-12), indicating that N-glycan processing can occur with this soluble reporter. FMK024 had no effect on recovery in the media fraction (lanes 13 and 14; Table 1). These data suggest that the overwhelming fate the GPI-minus reporter is degradation in the lysosomal compartment.
Localization of procyclin reporters in bloodstream trypanosomes
To determine the localization of the procyclin reporters in bloodstream parasites we performed IFA analysis. In the EPMH cells both anti-EP9 and anti-HA antibodies gave discrete and precisely overlapping signals in the flagellar pocket region just anterior of the kinetoplast (Fig. 4, panels A-D, arrowhead) validating both antibodies for detection of the reporter in bloodstream cells. The EPMH signal did not overlap to any degree with either the lumenal ER marker BiP (Fig. 4, panels E-H) or the lysosomal membrane marker p67 (Fig. 4, panels I-L). To confirm the site of EPMH localization as the flagellar pocket, live trypanosomes were exposed to biotinyl-tomato lectin at 5°C, conditions that allow external access to the lumen of the pocket, but which inhibit subsequent endocytosis (Langreth and Balber, 1975). Tomato lectin binds poly-N-acetyllactosamine-containing N-glycans on membrane glycoproteins in bloodstream trypanosomes, and hence is a specific marker for the flagellar pocket/endosomal system (Alexander et al., 2002
; Nolan et al., 1999
). After washing and fixation the cells were stained with anti-HA antibody to detect the reporter, and also with antibody to the paraflagellar rod, which extends the entire extracellular length of the flagellum but does not enter the cell body per se (Gull, 1999
). Anti-HA staining exactly overlaps the signal from extracellular tomato lectin at a site precisely between the end of the paraflagellar rod and the posterior kinetoplast (Fig. 4, panels M-P, arrowhead) unequivocally establishing the flagellar pocket as the steady state location for the GPI1 EPMH reporter.
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In cells expressing EPMHgpi the GPI0 reporter typically presented a reticular pattern of staining (Fig. 6; panels B,F,J) that co-localized predominantly with the ER marker BiP (panel D), but with some discrete staining in the lysosomal region (panel D, arrowhead), and modest co-localization with the lysosomal marker p67 (panel H, arrowhead). However, cells were occasionally observed in which more robust colocalization with p67 was evident (panel L, arrowhead). When cells were pretreated with FMK024 to block lysosomal degradation the reporter signal in the ER was diminished and elevated post-nuclear staining (Fig. 6, panels M-P) was seen in a region that also stained prominently for the lysosomal marker p67 (Fig. 6, panels Q-T). The co-localization of EPMH
gpi and p67 is not precise, rather it interdigitates as if the enlargement of the lysosomal compartment induced by FMK024 separates the signals deriving from membrane-bound p67 and the soluble reporter. We have seen a similar phenomenon when comparing the localization of endocytosed transferrin and p67 in FMK024-treated cells (Alexander et al., 2002
). It is not clear why the ER signal diminishes with FMK024 treatment (identical exposure times were used for all anti-EP9 images), but we have also seen a similar, albeit less pronounced, effect with GPI-minus VSG (Triggs and Bangs, 2003
). Overall these localizations are consistent with the pulse-chase data indicating that GPI0 EPMH
gpi is delivered to the lysosome where it is subject to degradation by endogenous protease activities
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In pulse chase analyses the BiPNHP reporter (Fig. 7A) was first detected as a discrete precursor (lane 1, 55 kDa) that was converted to a larger mature form during subsequent intracellular transport (lanes 2-5). Ultimately the mature form was released into the media with high efficiency (>80%, lanes 6-10). Treatment with FMK024 to block lysosomal degradation did not alter the pattern of processing or recovery of the mature form (Fig. 7B). BiP has no N-glycosylation sites, therefore it is likely that conversion to the mature form is due to GPI modification as we suggested above for the EPMH reporter. As is the case for EPMH, the nature of this GPI modification is unknown. These results support the observation made with EPMH that a single GPI anchor cannot prevent release from cells. However, since soluble (GPI0) BiPN is secreted from bloodstream trypanosomes, we wished to test this more rigorously with a reporter that is not normally secreted, p67. The p67HP reporter behaved in a manner qualitatively similar with that of the EPMH and BiPNHP reporters (Fig. 7C). It is initially detected as an
100 kDa glycoform (lane 1) and is processed to a larger mature form (lanes 2-5) that is subsequently released from cells (
15%, lanes 6-10). Processing to the mature form is due to extensive addition of pNAL to N-linked glycans (Alexander et al., 2002
). FMK024 treatment did not increase the fractional recovery of released p67HP, but the cell-associated fraction was increased indicating that most of the GPI-anchored reporter is still delivered to the lysosome (Fig. 7D). Collectively these results with exogenous reporters indicate that a single GPI anchor is sufficient to rescue secretory reporters, either in part or in full, from lysosomal targeting in bloodstream stage trypanosomes, but is not sufficient to prevent release from the cell surface.
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The level of endogenous TfR expression [2-3x103 molecules per cell (Steverding et al., 1995)] is relatively low compared with the expected levels of the various reporter constructs from our constitutive expression vector. To test if elevated expression of TfR would affect its relative distribution between lysosomal degradation and shedding we used transferrin starvation as a means to induce higher synthesis rates. Starvation can be achieved by culture in serum with low affinity transferrin ligand, e.g. canine serum, resulting in a
fivefold elevation in steady state TfR levels (Mussman et al., 2004
; Mussman et al., 2003
). We too find that culture (48 hours) in canine serum elicits an approximately sevenfold to tenfold increase in steady state TfR relative to cells cultured in bovine serum (estimated by immunoblotting of serially diluted cell extracts; data not shown). Supplementation with bovine holotransferrin greatly reduced this effect. Starved and control cells were subjected to pulse-chase analysis to determine the fate of up-regulated TfR (Fig. 8C). Transferrin starvation increased the de novo synthesis of both subunits 14-fold (compare lanes 1 and 5), and transferrin significantly reduced this effect (lane 9). However, the recovery of released TfR (T4 media fraction) did not increase as a percentage of initial synthesis (T0 cell fraction) in the induced cells (lanes 5 and 8, 2%) relative to control cells (lanes 1 and 4, 7%). Thus, increased synthesis does not result in more shedding, despite the fact that elevated TfR expression results in prominent surface localization (Fig. 9B,C) as was originally demonstrated by Mussman et al.
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Given our findings with the recombinant GPI1 reporters it seemed contradictory that increased surface expression would not also lead to increased release of the heterodimeric TfR. To test whether this surface receptor was actually functional, live cells were exposed to Tf:gold conjugates at 8°C to minimize endocytosis and binding was visualized by electron microscopy. Gold particles were found in the flagellar pocket and nearby endosomal elements in uninduced cells (Fig. 9D,G) and likewise to a much greater extent in transferrin-starved cells (Fig. 9E,H). However, under neither condition was binding seen on the external flagellum or cell surface. Competition with holotransferrin completely blocked Tf:gold binding (Fig. 9F,I). These results strongly suggest that surface TfR in starved cells is not functional heterodimer. A possible explanation for these seemingly contradictory results is proposed below.
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Discussion |
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Release of EPMH with an intact GPI anchor was initially surprising, however, there is precedence for desorption of GPI1 proteins from trypanosomal membranes. Ultrastructual studies show that TfR is dispersed within the lumen of the flagellar pocket, unlike VSG, which is intimately associated with the pocket membrane (Geuskens et al., 2000; Salmon et al., 1994
; Steverding et al., 1994
). The behaviors of both TfR and our GPI1 reporters are likely related to the exclusive use of myristate (C-14:0) in the diacylglycerol portion of the bloodstream form GPI anchor (Buxbaum et al., 1994
; Masterson et al., 1990
). The biological effect of this eccentricity is readily extrapolated from the desorption of free and macromolecule-bound phospholipids from model bilayer membranes. Dimyristoyl phosphatidylethanolamine desorbs from membranes with a rate (t1/2
14 minutes at 37°C) consistent with the extent of release observed for GPI1 reporters in our system, and furthermore, attachment of bulky hydrophilic head groups, e.g. apotransferrin, significantly enhances the rate of desorption (Silvius and Leventis, 1993
; Silvius and Zuckerman, 1993
). Lengthening the acyl chains by two (C-16:0, palmitoyl) or four (C-18:0, stearoyl) methylene groups reduces the PE desorption rate
60-fold and
1500-fold, respectively. It is logical then that the EPMH reporter is not shed from procyclic trypanosomes because this stage of the lifecycle synthesizes a more hydrophobic lysoacylglycerol (C-18:0), inositol acylated (mixed C-16:0 and C-18:0), GPI anchor (Treumann et al., 1997
), and also because the lower temperature (27°C) of procyclic cultures will decrease the desorption rate (Silvius and Leventis, 1993
). A strong biological imperative for why native VSG is a dimer can also be inferred. If bloodstream form trypanosomes have a surface glycocalyx composed of a single major GPI-anchored protein, and if that anchor is composed uniquely of dimyristoylglycerol, then dimerization is essential to increase the avidity of membrane association. Indeed, VSG is an extremely stable protein, turning over with a halftime in excess of 30 hours (Bulow et al., 1989
; Seyfang et al., 1990
), and yet low-level transfer of intact VSG to neighboring erythrocyte membranes can still be detected (Rifkin and Landsberger, 1990
). It is also worth noting that monovalent GPI-anchored lipophosphoglycan with a lysoacylglycerol chain length of C-24:0 to C-26:0 is also readily shed from the surface of the related kinetoplastid parasite Leishmania (Ilg et al., 1992
).
We propose a simple model for the general behavior of GPI1 proteins in bloodstream stage parasites based on the documented trafficking of VSG and endocytic cargo (Engstler et al., 2004; Grünfelder et al., 2003
; Overath and Engstler, 2004
). This model assumes that dissociation/reassociation from membranes can occur at any point during trafficking through the secretory/endocytic pathways, but dissociation at extracellular membranes is effectively irreversible because serum lipoproteins will act as a sink for monomer transfer (Silvius and Zuckerman, 1993
). A GPI1 reporter may arrive at the flagellar pocket in either a membrane-associated or soluble state. If membrane-associated it may diffuse laterally out onto the surface of the cell body, from which it will dissociate with a finite half-life. If already dissociated, it may then exit via the confined opening of the pocket. Alternatively, the GPI1 reporters can be endocytosed in either free or membrane-bound states and delivered to endosomal compartments for subsequent sorting. Reporters in the free state when they arrive at the Rab11+ recycling endosome are likely to be included in the Class II clathrin-coated vesicles that mediate trafficking to the late endosomal and lysosomal compartments as demonstrated for the fluid-phase markers, ferritin and horseradish peroxidase (Engstler et al., 2004
). GPI1 reporters that are membrane-bound at this point will be excluded from coated vesicles and returned by default to the flagellar pocket via exocytic carriers as shown for endogenous VSG (Overath and Engstler, 2004
). It is also possible that membrane association of internal GPI1 reporters is favored by the close proximity of enclosing membranes, and that GPI1 proteins are just not as effectively excluded from coated vesicles as is GPI2 VSG. In either case, recycling and sorting will continue until any single reporter molecule is either released into the media or delivered to the lysosome for degradation. An additional layer of cargo sorting may take place at the flagellar pocket, where soluble GPI1 reporters may be at a disadvantage for exit. There is little evidence for release of soluble secretory proteins in bloodstream stage parasites. Indeed, the only soluble reporter we have found to be effectively secreted is the truncated BiPN reporter (Triggs and Bangs, 2003
). Other soluble reporters, e.g. the lumenal domain of p67 or GPI-minus VSG, are primarily delivered to the lysosome (Alexander et al., 2002
; Triggs and Bangs, 2003
). Thus membrane attachment may facilitate lateral diffusion onto the cell surface, whereas soluble reporters are more prone to endocytic uptake. Another factor that may contribute to retention of soluble cargo is the poorly defined ground substance of the flagellar pocket lumen. The pocket is rich in extensive pNAL-bearing glycoconjugates (Nolan et al., 1999
) and it has been suggested that these glycans may form a `gel-like' matrix that could retard egress (Atrih et al., 2005
).
Our results are directly relevant to the recent finding that transferrin starvation induces upregulation of native TfR (ESAG6/ESAG7 heterodimer) expression, leading to spill-over of receptor onto the surface of bloodstream stage parasites (Mussman et al., 2004; Mussman et al., 2003
), a result that we have fully reproduced here. This was interpreted to indicate a saturable mechanism for TfR retention in the flagellar pocket, although no evidence for a `TfR receptor' exists. Nevertheless, a retention mechanism could explain why, of all our GPI1 reporters, native TfR is the least likely to be released from intact bloodstream trypanosomes. However, some explanation is also required for why spill-over does not lead to increased shedding of TfR from the cell surface, when our results so clearly indicate that a single GPI anchor will not maintain cell association. One possibility stems from the ability of both ESAG6 and ESAG7 to homodimerize when expressed independently (Salmon et al., 1994
). Nothing is known about the relative affinities for homotypic versus heterotypic subunit association, but native homodimerization cannot be excluded given that the two ESAGs are nearly identical in sequence. If so, then GPI0 ESAG7 homodimers should be delivered to the lysosome and degraded, as was observed for heterodimers of ESAG7 and GPI-minus ESAG6 (Biebinger et al., 2003
) and for GPI-minus VSG (Triggs and Bangs, 2003
). And, like native VSG, GPI2 ESAG6 homodimers should be stably cell-associated, which would account for surface localization without significant shedding into the media. Consistent with this explanation, and with the fact that ESAG6 homodimers are non-functional (Salmon et al., 1994
), we are unable to detect direct binding of Tf:gold to the cell surface of transferrin-starved cells, despite the presence of abundant immunoreactive material over the entire surface. Very similar results were presented by Mussman et al. (Mussman et al., 2003
), although they did detect low-level binding of biotinyl-Tf to the surface of transferrin-starved cells. However, specific binding was restricted to the flagellar pocket opening and the proximal membrane of the flagellum, and was conspicuously absent from the larger cell body. Collectively then, the lack of transferrin binding and the lack of TfR shedding strongly argue against the presence of significant amounts of functional GPI1 TfR heterodimers on the surface of transferrin-starved cells. Additional work will be required to confirm that ESAG6 homodimers are present on the cell surface.
In summary, our work confirms that GPI valence strongly influences post-Golgi trafficking of membrane proteins in bloodstream stage trypanosomes. Furthermore, these effects cannot be ignored in interpreting the behavior of native GPI1 proteins, i.e. procyclin and TfR, when expression is induced in this stage of the lifecycle. A future area that requires attention is the fate of additional reporters with a GPI2 valence and this work is already underway in our lab. Consequently our understanding of the role of GPI anchors in protein trafficking in these important pathogens should soon come into sharper focus.
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Acknowledgments |
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