Developmentally regulated trafficking of the lysosomal membrane protein p67 in Trypanosoma brucei

David L. Alexander*, Kevin J. Schwartz, Andrew E. Balber{ddagger} and James D. Bangs§

The Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, WI 53706, USA
* Present address: Department of Microbiology and Immunology, Stanford University Medical School, Fairchild Building, D305, Stanford, CA 94305, USA
{ddagger} Present address: StemCo Biomedical Inc., 2810 Meridian Parkway, Suite 148, Durham, NC 27713, USA

§ Author for correspondence (e-mail: jdbangs{at}facstaff.wisc.edu )

Accepted 28 May 2002


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
p67 is a lysosomal type I membrane glycoprotein of Trypanosoma brucei. In procyclic stage cells p67 trafficks to the lysosome without modification, but in the bloodstream stage Golgi processing adds poly-N-acetyllactosamine to N-glycans. In both stages proteolytic fragmentation occurs in the lysosome, but turnover is approximately nine times faster in bloodstream cells. Trafficking of wildtype p67 and mutants missing the cytoplasmic (p67{Delta}CD) or cytoplasmic/transmembrane domains (p67{Delta}TM) was monitored by pulse-chase, surface biotinylation and immunofluorescence. Overexpressed wildtype p67 trafficks normally in procyclics, but some leaks to the cell surface suggesting that the targeting machinery is saturable. p67{Delta}CD and p67{Delta}TM are delivered to the cell surface and secreted, respectively. The membrane/cytoplasmic domains function correctly in procyclic cells when fused to GFP indicating that these domains are sufficient for stage-specific lysosomal targeting. In contrast, p67 wildtype and deletion reporters are overwhelmingly targeted to the lysosome and degraded in bloodstream cells. These findings suggest that either redundant developmentally regulated targeting signals/machinery are operative in this stage or that the increased endocytic activity of bloodstream cells prevents export of the deletion reporters.

Key words: Trypanosome, Lysosome, Flagellar pocket, Endocytosis, LAMP


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
African trypanosomes are parasitic protozoa that cause human (sleeping sickness) and veterinary (Nagana) disease throughout the sub-Saharan continent. Phylogenetically, trypanosomes represent a deeply rooted branch of the eukaryotic kingdom (Sogin et al., 1989Go) and consequently provide a novel model system for studying basic eukaryotic processes. The lifecycle of these parasites alternates between the bloodstream form in mammalian hosts and the procyclic form in the insect vector, the tsetse fly. Trypanosomes are highly polarized cells with an elongated morphology (reviewed by Balber, 1990Go; Bangs, 1998Go). A single flagellum emerges from a posterior invagination of the plasma membrane, the flagellar pocket, and adheres along the cell body in the anterior direction. Owing to the close packing of subpellicular microtubules beneath all other parts of the plasma membrane, the flagellar pocket is the only external membrane that is available for vesicular trafficking and, insofar as it is known, all export and import of macromolecules takes place at this domain (Webster and Russell, 1993Go). Consequently, the flagellar pocket represents a unique intersection of the secretory and endocytic pathways in this unusual cell. Since it is essential for the bloodstream parasite to both export the major coat protein, variant surface glycoprotein, and import host-derived nutritional macromolecules, such as transferrin, the flagellar pocket must play a pivotal role in the pathogenesis associated with trypanosomiasis.

Between the flagellar pocket and the central nucleus reside many membranous compartments involved in these trafficking pathways, and a growing number of specific protein markers have been defined. These include a homologue of the ER molecular chaperone BiP (Bangs et al., 1993Go); Rab homologues (Field et al., 2000Go); a clathrin homologue (Morgan et al., 2001Go); and an unusual glycosylphosphatidylinositol-anchored heterodimeric transferrin receptor in the flagellar pocket (Ligtenberg et al., 1994Go; Salmon et al., 1994Go). There are two markers for the terminal lysosomal compartment, trypanopain, the major soluble thiol protease of trypanosomes (Caffery et al., 2001Go; Mbawa et al., 1991Go), and p67, a membrane glycoprotein (Brickman and Balber, 1993Go; Kelley et al., 1999Go).

Originally called CB1-gp, p67 was first identified as the ligand for an N-glycan-specific monoclonal antibody (CB1) generated using total bloodstream trypanosome ricin-binding proteins as immunogen (Brickman and Balber, 1993Go). p67 has a type I topology (Fig. 1A) with a highly glycosylated N-terminal domain, a 19-residue transmembrane domain and a 24-residue C-terminal cytoplasmic domain (Kelley et al., 1999Go). It bears a striking resemblance to mammalian lysosome-associated membrane proteins (LAMPs), although there are no sequence homologies. In both stages p67 is synthesized as a 100 kDa N-glycosylated ER species (gp100), and in bloodstream trypanosomes N-glycan processing in the Golgi generates the CB1 epitope converting gp100 to gp150. Thereafter it is delivered to the lysosome, at least in part, by export to the flagellar pocket followed by endocytosis (Brickman and Balber, 1994Go). In procyclic trypanosomes p67 N-glycans are not processed and delivery to the lysosome is direct from the Golgi (Kelley et al., 1995Go). Thus there are several stage-specific aspects to both the processing and the trafficking of p67.



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Fig. 1. Vectors, peptide map and reporter constructs. (A) p67 processing events. The deduced topological structure (top) and the positions of the various glycoforms (bottom) are indicated. N-termini determined by direct peptide microsequencing are numbered by codon position within the deduced open reading frame. Black boxes, hydrophobic N-terminal signal sequence and C-terminal transmembrane domain; hatched box, glycosylated lumenal domain; open box, C-terminal cytoplasmic domain; lollipops, consensus N-glycosylation sites; dashed lines, uncertain cleavage sites. (B) Deduced amino acid sequence of the p67 transmembrane (black box) and C-terminal cytoplasmic domains. Acidic motifs are underlined and di-leucine motifs are in large font. The position of stop codons engineered to create the p67{Delta}CD and p67{Delta}TM reporter constructs are indicated. (C) Diagram of GFP fusion reporters. Black boxes, EP1 procyclin signal sequence and p67 transmembrane domain; gray box, EGFP orf; open box, p67 cytoplasmic domain. (D) The pXS5 stable expression vector. Segments from KpnI to SacI are labeled as described in Materials and Methods. Restriction sites are: K, KpnI; X, XhoI; C, ClaI; H, HindIII; R, EcoRI; S, SmaI; A, AscI; P, PacI, SI, SacI. Bar, 500 bp.

 

Significant questions remain concerning the structure of p67, its trafficking and turnover, and its subcellular localization. For instance, bloodstream form p67 is cleaved into discrete fragments and degraded upon arrival in the lysosome, but the fate of p67 in procyclic cells is unclear. More interestingly, the signals that mediate stage-specific targeting of p67 remain to be elucidated. By analogy to mammalian LAMPs such signals would be predicted to reside in the cytoplasmic domain. In this work we present a detailed kinetic analysis of the biosynthesis, transport and turnover of p67, including an ordered map of proteolytic cleavages that occur in the lysosome in both stages of the life cycle. In addition, we define p67 localization in relationship to other known markers of the secretory and endocytic pathways. Using this information we then investigate the targeting of p67 by expression of C-terminal truncation mutants in both bloodstream and procyclic trypanosomes. Our results indicate that while lysosomal targeting in procyclic cells occurs in a predictable manner, targeting in bloodstream cells is more complex and may involve novel stage-specific mechanisms.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Parasite culture and radiolabeling
Trypanosoma brucei Lister 427 strain trypanosomes were used in all experiments. Procyclic cell lines were maintained at 27°C in Cunningham's medium with 15% inactivated FBS (Cunningham, 1977Go). MITat 1.4 bloodstream cell lines were maintained in HMI9 medium at 37°C in humidified 5% CO2 (Hirumi and Hirumi, 1994Go). Pulse-chase radiolabeling experiments were performed with [35S]methionine/cysteine as described previously (Bangs et al., 1996Go; Bangs et al., 1997Go). In experiments with the thiol protease inhibitor P27 (morpholinourea-phenylalanine-homophenylalanine-benz-{alpha}-pyrone, a generous gift of James McKerrow, University of California-San Francisco) bloodstream cells were preincubated with 2 µM compound for 1 hour and then throughout all subsequent in vitro manipulations.

Immunological procedures
Rabbit anti-TbBiP, anti-TbHSP70.4 and anti-VSG antibodies have been described previously (Bangs et al., 1996Go; Bangs et al., 1993Go; McDowell et al., 1998Go). Rabbit antibody to the p67 cytoplasmic domain (anti-CD) was generated by immunization with a synthetic peptide (KTEEDLLPEEAEGLIDPQN) coupled to keyhole limpet hemocyanin. Monoclonal anti-p67 (mAb280), which recognizes and uncharacterized peptide epitope in the p67 lumenal domain, was a generous gift of David Russell (Cornell University, Ithica, NY). Rabbit anti-tomato lectin antibody was generated by immunization with purified lectin (Vector Laboratories, Burlingame CA). Rabbit anti-T. brucei transferrin receptor antibody was a generous gift of Piet Borst (Netherlands Cancer Institute, Amsterdam) and rabbit antirhodesain (trypanopain) was a generous gift of Conor Caffrey (University of California-San Francisco). Rabbit anti-GFP was a generous gift of Gerard Marriott (University of Wisonsin-Madison).

Standard immunoprecipitations, surface biotinylation and streptavidin blotting were carried out as described previously (Bangs et al., 1997Go). Gels and blots were analyzed by phosphorimaging and/or chemiluminescence and final images were processed in PhotoShop 5.5. For pulse-chase analyses of the p67{Delta}CD and p67{Delta}TM reporters extracts of transgenic cell lines were cycled through three rounds of immunoprecipitation with anti-CD to remove all endogenous full-length p67 polypeptides (D.L.A. and J.D.B., unpublished). The remaining p67 reporter polypeptides were then immunoprecipitated with mAb280. Enzymatic deglycosylation of immunoprecipitates with peptide:N-glycosidase F was performed according to manufacturer's instructions (New England Biolabs, Beverly, MA).

Microsequencing
Total membranes of hypotonically lysed MITat 1.4 bloodstream trypanosomes (2x1010) were extracted in TEN buffer (50 mM TrisHCl, pH 7.5, 150 mM NaCl, 5 mM EDTA) containing detergents (1% NP-40, 0.5% deoxycholate, 0.1% SDS) and protease inhibitors (0.1 mM tosyllysine chloromethyl ketone, 0.1 mM PMSF, 2 µg/ml each of leupeptin, antipain, chymostatin and pepstatin). p67 polypeptides were immunoselected with an mAb280 IgG column. 600 µg was fractionated by SDS-PAGE and electrotransfered to PVDF membranes for microsequencing (Midwest Analytical, St Louis, MO).

Reporter constructs
All reporter constructs were prepared by PCR. Truncated p67 reporters were generated by placing stop codons before or after the transmembrane domain (Fig. 1B). Secretory EGFP reporters (Fig. 1C) were prepared by in frame fusion of the EP1 procyclin signal sequence (codons 1-38), EGFP (codons 1-239; Clontech), and the transmembrane and/or cytoplasmic domains of p67 (codons 617-639 or codons 617-659). All reporter constructs were cloned into the HindIII/EcoRI sites of the trypanosomal stable expression vectors, pXS2neo (Bangs et al., 1996Go) and pXS5neo for stable transformation of procyclic and bloodstream trypanosomes, respectively. pXS5neo is a derivative of pXS2 containing (5'-3', Fig. 1D): 403 bp from the untranscribed 3' end of the rDNA locus; a 487 bp rRNA promoter element (nts -260 to 227 relative to initiation); a 78 bp splice acceptor fragment (nt -87 to -10 relative to the EP1 procyclin start codon); a multicloning region; a modified 871 bp intergenic region (nt 22 3' of the aldolase ORF to nt 12 of the downstream orf); the neomycin phosphotransferase gene; the ß{alpha}-tubulin intragenic region (nts -108 to +532 bp relative to the ß-tubulin stop codon). Linearized vectors (pXS2, BstXI; pXS5, XhoI) were introduced into cultured procyclic and bloodstream trypanosomes by electroporation and stable transformants were selected with neomycin.

Uptake studies
Cultured log phase MITat 1.4 bloodstream trypanosomes were washed, resuspended (5x107/ml) in serum-free HMI9 medium containing 1% bovine serum albumin, and preincubated (1 hour, 37°C, 5% CO2). Incubation was continued for 1 hour in the presence of Alexa488-conjugated transferrin (5 ug/ml, Molecular Probes, Eugene OR) or biotinyl-tomato lectin (5 µg/ml, Vector Laboratories). For transferrin uptake P27 (2 µM) was included in both incubations to inhibit degradation by lysosomal thiol protease. As a control for binding specificity, chitin hydrosylate (1/1000 final dilution, Vector Laboratories) was included in duplicate tomato lectin samples. In all experiments cell viability remained excellent. Following uptake, cells were washed and processed for immunofluorescence.

Microscopy
Fixed permeabilized procyclic cells were prepared for immunofluorescence as described previously (Roggy and Bangs, 1999Go). Cultured bloodstream cells were washed in ice cold PBS with 10 mg/ml glucose, fixed lightly (107 cells/ml, 0.1% formaldehyde in PBS, 5 minutes, 4°C), centrifuged and resuspended (4x107 cells/ml) in PBS with 5% normal goat serum (NGS). Fixed cells (50 µl) were smeared on prewashed slides, air dried, extracted sequentially with methanol and acetone (3 minutes each, -20°C), and then stained as with procyclic cells. Specific staining was developed with appropriate Alexa488- or Alexa633-conjugated secondary antibodies, or Alexa488-streptavidin (Molecular Probes), with 500 ng/ml DAPI. Serial 0.2 µm image Z-stacks were collected at 100x on a motorized Zeiss Axioplan IIi equipped with a rear-mounted excitation filter wheel and a triple pass (DAPI/FITC/Texas Red) emission cube. Fluorescence images were captured with a Zeiss AxioCam B&W CCD camera and were lightly deconvolved by a nearest neighbor algorithm, psuedocolored, and merged using OpenLabs 3.0 software (Improvision, Lexington, MA).


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stage-specific processing of p67
Pulse-chase radiolabeling experiments were performed to investigate stage-specific post-translational processing of p67 (Fig. 2). As previously reported (Brickman and Balber, 1994Go; Kelley et al., 1999Go), p67 is synthesized in bloodstream cells as an ER-associated gp100 glycoform and is converted to the gp150 glycoform by N-glycan processing (Fig. 2A). The gp150 glycoforms binds poly-N-acetyllactosamine (pNAL)-specific tomato lectin [(Nolan et al., 1999Go) D.L.A. and J.D.B., unpublished)]. Thereafter p67 is delivered to the lysosome, where proteolytic fragmentation generates discrete gp75, gp42, gp32 and gp28 glycoforms (Fig. 2A,D). The gp28 species is only observed in overexposures of prolonged pulse radiolabelings (D.L.A. and J.D.B., unpublished). Fragmentation of p67 is almost completely blocked by P27 (Fig. 2B), a potent inhibitor of the major lysosomal thiol protease, trypanopain (Caffery et al., 2001Go; Mutomba and Wang, 1998Go). Extended pulse-chase analyses reveal for the first time that p67 is also subject to proteolytic fragmentation in procyclic cells (Fig. 2C,D). As in bloodstream cells, p67 is first detected as the gp100 glycoform but subsequent N-glycan processing does not occur. During the chase period p67 is reduced to glycoforms of similar masses to those seen in bloodstream cells. p67 turnover in procyclics is also impaired by the inhibitor P27 (D.L.A. and J.D.B., unpublished). The rates of turnover as measured by disappearance of gp100 are t1/2 0.7±0.1 and 6.0±0.5 hours, respectively, for bloodstream and procyclic cells (Table 1). For procyclics this rate represents delivery and subsequent degradation in the lysosome. For bloodstream cells it represents delivery to the Golgi where conversion to gp150 occurs. This interpretation is supported by the accumulation of gp150 seen in bloodstream cells when turnover is inhibited with P27 (Fig. 2B).



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Fig. 2. Processing of p67. (A-C) Pulse-chase analyses. Bloodstream (A,B) and procyclic (C) trypanosomes were pulse radiolabeled for 15 minutes and then chased in the absence (-) or presence (+) of the thiol protease inhibitor P27. At the indicated chase times p67 polypeptides were immunoprecipitated from cell extracts with mAb280 and analyzed by SDS-PAGE/fluorography. All lanes contain 107 cell equivalents. The mobilities of molecular mass markers in kDa and the positions of the various p67 glycoforms are indicated. (D) Procyclic (Pro) and bloodstream (BS) trypanosomes were metabolically radiolabeled for 9 and 4 hours, respectively, and p67 polypeptides were immunoprecipitated with mAb280. Immunoprecipitates were mock-treated (-) or PNG-treated (+) to remove N-glycans and analyzed by SDS-PAGE/fluorography. The positions of the native glycoforms (gp150 to gp32) and of the corresponding deglycosylated peptides (d150 to d32) are indicated. The origin of the smallest deglycosylated bloodstream polypeptide (?) is not known. All lanes contain ~107 cell equivalents.

 

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Table 1. Properties of p67 reporters in transformed trypanosome cell lines

 

To confirm that all p67 glycoforms are encoded in the same open reading frame, steady state p67 polypeptides were immunoaffinity purified from bloodstream trypanosomes for N-terminal microsequencing. Peptide sequences were obtained for gp32 (DATPTVVTVW) and gp42 (SAFVKVVKDD) that unambiguously indicate cleavages at codons 38 and 241 of the deduced p67 amino acid sequence. The signal sequence cleavage site (codon 35) of mature gp100 was previously determined with procyclic p67 (Kelley et al., 1995Go). In addition, fragments bearing an intact C-terminus (gp150, gp100, gp75 and gp28) were identified by reactivity with antipeptide antibody specific for the cytoplasmic domain (anti-CD; D.L.A. and J.D.B., unpublished). Collectively these data indicate an ordered map for p67 proteolytic processing (Fig. 1A) and confirm that all glycoforms are derived from a full-length p67 precursor polypeptide.

The patterns of p67 fragmentation in both stages of the lifecycle are strikingly similar given that the full-length precursor glycoforms differ by ~50 kDa. To investigate this issue radiolabeled p67 from procyclic and bloodstream cells were treated with peptide:N-glycosidase F (PNG) to remove N-glycans. With the exception of gp150, which is absent in procyclic cells, the sizes of the native glycoforms are closely matched in both stages of the lifecycle (Fig. 2D, compare lanes 1 and 3). Furthermore, PNG treatment generates a similar set of deglycosylated polypeptides (dp150 to dp32, compare lanes 2 and 4). As previously demonstrated (Kelley et al., 1995Go), deglycosylation reduces the full-length glycoforms (gp150 and gp100) to a single 67 kDa species (d150/d100). Assignment of the deglycosylated gp75 and gp42 species (d75 and d42) is presumed by relative shifts in electrophoretic mobility. The identical profiles of deglycosylated polypeptides indicate that cleavage site selection is not stage-specific. Furthermore, the size similarity of the native fragments in each stage suggest that the terminal N-glycan modifications that generate the bloodstream-specific gp150 glycoform are removed rapidly upon arrival in the lysosome, likely concomitant with proteolytic fragmentation.

Subcellular localization of p67
The steady-state subcellular localization of p67 was investigated by immunofluorescence with mAb280 to detect p67 and anti-BiP as a marker for the ER (Fig. 3). In both procyclic (Fig. 3E) and bloodstream trypanosomes (Fig. 3F) BiP localizes to a characteristic network of the ER (green) indicating good preservation of morphology (Bangs et al., 1993Go). In each case, p67 localizes to a prominent vesicular compartment (red) immediately posterior to the nucleus and well forward of the flagellar pocket. This pattern is typical, although p67 sometimes presents as multiple discrete vesicles in the same region (Fig. 3H). The p67-positive region also stains for trypanopain indicating that it is a hydrolytic compartment (Fig. 3G, yellow, arrowhead). The distribution of p67 relative to total tomato lectin-reactive polypeptides in bloodstream cells was also assessed (Fig. 3H). Consistent with previous work (Nolan et al., 1999Go), tomato lectin detects pNAL-bearing glycoconjugates (green) throughout the post-nuclear region, which contains many components of the secretory/endocytic pathways. p67 discretely co-localizes with a subset of the tomato lectin staining compartments (Fig. 3H, arrowheads).



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Fig. 3. Localization of p67. Procyclic (A,E) and bloodstream (B and F, C and G, D and H) trypanosomes were fixed and permeabilized as described in Materials and Methods. Cells were stained with monoclonal mAb280 anti-p67 (E-H, red) and either rabbit anti-BiP (E,F, green), rabbit anti-trypanopain (G, green) or biotinyl-tomato lectin (H, green). Staining was visualized with appropriate secondary reagents and all samples were counterstained with DAPI (blue). Merged DIC and DAPI images (A-D) reveal the positioning of the large central nucleus (n) and compact posterior kinetoplast (k). Corresponding merged three channel fluorescent images are presented (E-H). Regions of colocalization appear yellow in merged images, and the matched single channel red (left) and green (right) images in the region of the lysosome are inset below for G and H. Arrowheads indicate regions of precise p67 co-localization with trypanopain and tomato lectin in G and H.

 

Immunoelectron microscopy studies suggest that p67 is a marker for the terminal endocytic compartment in trypanosomes (Brickman and Balber, 1993Go; Brickman et al., 1995Go). We have confirmed this by immunofluorescent localization of p67 following uptake of either fluorescent transferrin or biotinyl-tomato lectin as receptor-mediated cargo in intact bloodstream trypanosomes (Fig. 4). Endocytosed transferrin is rapidly degraded in bloodstream trypanosomes (Grab et al., 1992Go; Steverding et al., 1995Go) and its intracellular detection is dependent on the presence of trypanopain inhibitors. Even short exposure (2 hours) to such inhibitors results in significant lysosomal swelling (Fig. 4B,D, compare red inserts). Consequently, endocytosed transferrin (Fig. 4B, green insert), which is delivered to the lumen of the lysosome, appears to have a halo of membrane-bound p67 in the merged image. In contrast, endocytosed tomato lectin (Fig. 4D) is relatively resistant to degradation, no protease inhibitor is required, and the patterns of localization for p67 (red insert) and tomato lectin (green insert) are completely superimposable in the merged image. The receptor(s) for endocytosis of tomato lectin is not known, but many endogenous proteins in the flagellar pocket, including transferrin receptor, have pNAL-containing N-glycans that could serve as lectin-binding sites (Nolan et al., 1999Go). Whatever the receptor(s), inclusion of chitin hydrosylate blocks uptake of tomato lectin (Fig. 4F). These data clearly demonstrate that endocytosed macromolecules are targeted to, and degraded in, a compartment for which p67 is a definitive steady state marker.



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Fig. 4. Endocytosis of receptor-mediated cargo. Bloodstream trypanosomes were incubated (1 hour, 37°C) with Alexa488-conjugated transferrin (A,B), biotinyl-tomato lectin (C,D), or biotinyl-tomato lectin and chitin hydrolysate (E,F) as described in Materials and Methods. Transferrin uptake was performed in the presence of 2 µM P27 to prevent degradation. Cells were then fixed, permeabilized and processed for fluorescence microscopy. Merged DIC/DAPI images (A,C,E) and corresponding merged three color fluorescent images (B,D,F) are presented (blue, DNA; red, p67; green, ligand). Regions of colocalization appear yellow in merged images and the matched single channel images of p67 (left) or ligand (right) staining in the region of the lysosome are inset at the bottom for each three channel image.

 

Generation of truncated p67 reporters
To test our hypothesis that the signals mediating proper lysosomal targeting reside in the p67 cytoplasmic domain, constructs were engineered that placed stop codons immediately before or after the transmembrane domain (Fig. 1B). These reporters should either be membrane-associated (p67{Delta}CD) or soluble (p67{Delta}TM), and in both cases would be predicted to be defective in lysosomal targeting if the relevant signals reside in the cytoplasmic tail. Transgenic procyclic and bloodstream cell lines stably expressing these reporters, as well as control cell lines overexpressing wildtype p67 (p67WT), were prepared and analyzed by three methodologies, pulsechase radiolabeling (Fig. 5), cell surface biotinylation (Fig. 6), and immunofluorescence (Fig. 7). In the pulse-chase analyses endogenous p67 polypeptides were first removed from cell lysates with anti-p67 cytoplasmic domain (anti-CD) before specific immunoprecipitation of the truncation reporters with mAb280.



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Fig. 5. Biosynthesis and turnover of p67 reporter constructs. Procyclic (A,C) and bloodstream (B,D) cell lines stably expressing the truncated reporters, p67{Delta}CD (A,B) and p67{Delta}TM (C,D), were pulse radiolabeled (15 minutes) and then chased. At the designated times, samples (107 cell equivalents) were separated into cell and medium fractions and endogenous p67 polypeptides were removed by three rounds of immunoprecipitation with anti-CD antibody. p67 reporter polypeptides were then immunoprecipitated with mAb280 and analyzed as in Fig. 2. Panels A, B and D were generated by phosphorimaging and panel C by fluorography. Positions of the various p67 glycoforms are indicated.

 


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Fig. 6. Surface biotinylation of p67 reporter constructs. Untransformed (control) and transgenic (p67{Delta}CD or p67WT) procyclic (A) and bloodstream (B) cell lines were surface biotinylated and cell lysates were subjected to specific immunoprecipitation with mAb280 anti-p67 (P), anti-hsp70 (H), and anti-transferrin receptor (T) antibodies. Immunoprecipitates were fractionated by SDS-PAGE, electrotransfered to membranes, blotted with HRP-strepavidin, and developed by chemiluminescence. All lanes contain 107 cell equivalents. Scale represents relative molecular mass in kDa.

 


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Fig. 7. Immunolocalization of p67 reporters. Transgenic procyclic (A-D) and bloodstream (E-H) cell lines expressing wildtype p67 (WT) or p67{Delta}CD ({Delta}CD) were fixed/permeabilized and immunostained with mAb280 to detect total p67 polypeptides (red). Cells were counterstained with DAPI (blue) to reveal nuclei and kinetoplasts. Matched sets of digitally merged DIC/DAPI and anti-p67/DAPI images are presented. All images were deconvolved and processed identically.

 

p67 targeting in procyclic trypanosomes
Pulse-chase analyses in transgenic procyclic cells confirm the expected behaviors for the truncation reporters. p67{Delta}CD is synthesized as a gp100 glycoform that remains cell-associated throughout the 8 hour chase period (Fig. 6A; 78±6%, n=4). Little proteolytic fragmentation is apparent and no reporter is detected in the extracellular medium (D.L.A. and J.D.B., unpublished). Indeed, p67{Delta}CD is very stable in procyclic cells (t1/2>18 hours; Table 1). The p67{Delta}TM reporter is also synthesized as a gp100 glycoform in procyclic cells and is largely secreted to the media (Fig. 5C; 85±6%, 8 hours, n=6) with a rate (t1/2 4.0±0.7 hours; Table 1) that is within the range of other soluble reporters in procyclic trypanosomes (t1/2 1-5 hours) (Bangs et al., 1996Go; Bangs et al., 1997Go). Pulse-chase analyses of procyclic cells overexpressing wildtype p67 (D.L.A. and J.D.B., unpublished) revealed no significant alteration in the rate of turnover relative to that of endogenous p67 in untransformed cells (Table 1).

The localization of the p67 reporters in procyclic cells was investigated by cell surface biotinylation (Fig. 6A). Cells remained fully viable throughout the biotinylation procedure and the cytosolic marker Hsp70 was used as a control for cell integrity. Little or no biotinylated Hsp70 is detected in any procyclic cell line (Fig. 6A, lanes 2,4,6) indicating that plasma membrane integrity was maintained throughout the biotinylation procedure. In untransformed procyclic cells no p67 polypeptides are detected on the cell surface (Fig. 6A, lane 1). In contrast, cell surface p67 polypeptides are readily detected in transgenic procyclics expressing either the p67{Delta}CD (Fig. 6A, lane 3) or p67WT (Fig. 6A, lane 5) reporters. Consistent with the pulse-chase results, biotinyl-p67{Delta}CD remains cell-associated for >24 hours (D.L.A. and J.D.B., unpublished).

These findings were directly confirmed by immunofluorescent localization (Fig. 7). In addition to the typical internal staining pattern for endogenous p67 polypeptides, a distinct halo of cell surface staining is seen in the p67{Delta}CD cell line (Fig. 7D) and a faint, but reproducible, pattern of surface staining is also seen in p67WT cells (Fig. 7B). No cell surface staining is ever detected in cells expressing p67{Delta}TM or in untransformed control cells (D.L.A. and J.D.B., unpublished). Collectively these results argue that the signal(s) for proper targeting of p67 in procyclic trypanosomes reside in the cytoplasmic domain. Deletion of this domain leads to stable cell surface localization and additional deletion of the transmembrane domain leads to quantitative secretion. Furthermore, the existence of a minor pool of cell surface p67 when the full-length wildtype gene is overexpressed suggests that the machinery for targeting in procyclic cells can be saturated.

p67 targeting in bloodstream trypanosomes
Striking differences in behavior were found when the various reporters were analyzed in transgenic bloodstream trypanosomes. As expected, p67{Delta}CD is synthesized as a gp100 glycoform and is processed to the corresponding gp150 glycoform (Fig. 5B). Surprisingly, however, this reporter was then fragmented to the smaller glycoforms characteristic of delivery to the lysosome. Consistent with this interpretation, degradation is blocked with the thiol protease inhibitor P27 (D.L.A. and J.D.B., unpublished). Turnover of p67{Delta}CD in bloodstream cells is essentially the same as that of endogenous wildtype p67 (t1/2 0.6±0.1 hour; Table 1). The p67{Delta}TM reporter is also synthesized as gp100 and processed to gp150, but very little is actually secreted (Fig. 5D; 5.3±2.3%, at 4 hours, n=6). Most of this reporter is degraded internally with a turnover rate identical to that of endogenous wildtype p67 (t1/2 0.7±0.2 hours; Table 1), and P27 blocked degradation leading to the accumulation of the gp150 glycoform with no increase in secretion (5.1±2.4% at 4 hours, n=6). Overexpression of the wildtype p67 gene has no measurable effect on the normal turnover of total p67 polypeptides (t1/2 0.7±0.1 hours; Table 1).

Biotinylation assays were performed to detect the presence of these reporters on the surface of transgenic bloodstream cells (Fig. 6B). As with procyclic cells, controls for cell integrity were performed with hsp70 (Fig. 6B, lanes 2,5,8), and again, no biotinyl-hsp70 is detected. The bloodstream-specific transferrin receptor was also used as a positive control for biotinylation of proteins within the extracellular lumen of the flagellar pocket. In all cases a robust signal is evident for the mature ~60 kDa and ~42 kDa subunits of this receptor (Fig. 6B, lanes 3,6,9) indicating that the relatively sequestered lumen of the flagellar pocket does not restrict access of the external biotinylation reagent. No biotinylated p67 polypeptides are detected on the surface of either control untransformed cells (Fig. 6B, lane 1) or on cells overexpressing wildtype p67 (Fig. 6B, lane 7), but cell surface p67 is consistently present in the p67{Delta}CD cell line (Fig. 6B, lane 4). The presence of a surface pool of p67{Delta}CD was confirmed by immunofluorescence (Fig. 7H). No surface fluorescence is seen on bloodstream cells overexpressing wildtype p67 (Fig. 7F) or on control and p67{Delta}TM expressing cells (D.L.A. and J.D.B., unpublished). These results differ dramatically from those obtained with the procyclic cell lines. Low levels of p67{Delta}TM and p67{Delta}CD do escape to the medium and cell surface, respectively, indicating that the cytoplasmic domain contributes to proper targeting in bloodstream trypanosomes. However, the overwhelming fate of these reporters is degradation by a P27-inhibitable protease suggesting that lysosomal delivery continues in the absence of the signal(s) that are required for targeting in procyclic cells.

Targeting GFP to the lysosome
To determine whether signals in the p67 transmembrane and/or cytoplasmic domains are sufficient for lysosomal targeting in procyclic cells we fused a secretory form of green fluorescent protein (GFP) to the transmembrane domain alone (sGFP{Delta}CD) or to both domains together (sGFPWT) (Fig. 1C). In pulse-chase analyses radiolabeled polypeptides of the expected mass are detected at equivalent levels in each stable cell line (Fig. 8, compare lanes 1 and 5). At the end of the chase period recovery of the sGFP{Delta}CD reporter is the same in the absence (Fig. 8, lane 2, 52±6.5%) and presence (Fig. 8, lane 4, 56±8.6%) of trypanopain inhibitor. Recovery of the sGFPWT reporter is significantly lower (Fig. 8, lane 6, 24±7.3%, P<0.05), but this loss is reversed by inhibition of trypanopain (Fig. 8, lane 8, 67±13%, P<0.05). Biotinylation experiments also reveal both reporters at the cell surface with sGFP{Delta}CD being considerably more abundent than sGFPWT (K.J.S. and J.D.B., unpublished). The subcellular localization of the GFP reporters was investigated by immunofluorescence (Fig. 9). In both cell lines steady state accumulation of GFP in the ER and nuclear envelope is consistently observed, as shown by intense colocalization with BiP (Fig. 9E,G, yellow). The slow kinetics of GFP folding likely results in ER retention by quality control machinery (Hammond and Helenius, 1995Go; Reid and Flynn, 1997Go). In the sGFPWT cell line prominent GFP staining is also seen in a non-ER post-nuclear region (Fig. 9G, green, arrowhead), which is confirmed as the lysosome by robust colocalization of GFP with p67 (Fig. 9H, yellow, arrowhead). No such colocalization is observed with sGFP{Delta}CD (Fig. 9F, red, arrowhead). Collectively these results are entirely consistent with the behavior of the corresponding p67WT and p67{Delta}CD reporters and suggest that the p67 cytoplasmic domain targets heterologous reporters to the lysosome in procyclic trypanosomes.



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Fig. 8. Expression of GFP reporters. (A) Stable cell lines expressing sGFP{Delta}CD or sGFPWT were pulsed radiolabeled (15 minutes) in the absence (-) or presence (+) of trypanpain inhibitor and cell extracts were prepared at the indicated chase times. Reporter polypeptides were immunoprecipitated with anti-GFP and analyzed by phosphorimaging following SDS-PAGE. For quantitation the experiment was performed in triplicate and a representative image is presented. All lanes contain 5x106 cell equivalents.

 


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Fig. 9. Targeting of GFP reporters. Fixed/permeabilized procyclic cell lines expressing sGFP{Delta}CD ({Delta}CD) or sGFPWT (WT) were costained with specific antibodies for GFP (E-H, green) and BiP (E,G, red) or p67 (F,H, red). Cells were counterstained with DAPI (blue) to reveal nuclei and kinetoplasts. Matched DIC/DAPI (top) and three channel merged immunofluorescence (bottom) images are presented. Arrowheads indicate lysosomal regions of interest as discussed in the text. Single channel red (left) and green (right) images of the lysosomal region of each cell are inset in the bottom panels.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transport and processing of endogenous p67
In both procyclic and bloodstream trypanosomes p67 localizes to a prominent compartment between the central nucleus and the posterior flagellar pocket. Typically this is a single irregular vacuole but it occasionally presents as several closely associated vesicles. This p67-positive compartment is the primary intracellular site of the major lysosomal thiol protease trypanopain, and is also the ultimate repository in bloodstream trypanosomes for endocytic ligands such as transferrin and tomato lectin. In addition, trypanosome lytic factor, a toxic subclass of human HDL that is endocytosed via the flagellar pocket, is delivered to this terminal p67-containing compartment (Shimamura et al., 2001Go). These observations establish p67 as a definitive marker for the terminal lysosomal compartment of these important protozoan parasites, and are consistent with electron microscopy studies (Brickman and Balber, 1993Go; Brickman et al., 1995Go; Kelley et al., 1995Go; Lingnau et al., 1999Go), which also found p67 in pre-lysosomal compartments.

The fate of p67 was originally characterized in bloodstream cells and we have now extended these studies to the procyclic stage. In bloodstream cells essentially all ER gp100 is processed to gp150 by N-glycan modifications in the Golgi. Thus the disappearance of gp100 represents the rate of transport to the Golgi. However, subsequent transport to the lysosome must be rapid because p67 fragments can be detected before complete loss of the gp100 precursor. In procyclic trypanosomes, 100 kDa p67 is transported without N-glycan modification to the lysosome where it is also fragmented, probably by trypanopain. The disappearance of gp100 in this stage represents both the rates of transport to the lysosome and subsequent proteolysis. Consequently, the measured halftime probably overestimates the time required for transport. Developmental downregulation of trypanopain activity in the procyclic stage (Caffery et al., 2001Go) probably contributes to the slower rate of p67 turnover, as does the lower growth temperature of procyclics (27°C vs 37°C). Nevertheless, transport of p67 to the lysosome in procyclic cells is clearly slower than in bloodstream cells, which explains why previously no degradation of p67 was detected at shorter chase times in procyclics (Kelley et al., 1995Go).

Available evidence suggests that processing of gp100 to gp150, which generates the CB1 epitope, involves addition of terminal poly-N-acetyllactosamine (pNAL) to core N-glycans (Brickman and Balber, 1993Go; Nolan et al., 1999Go). However, addition of pNAL cannot fully account for the maturation of gp150 N-glycans. Many proteins of the bloodstream endocytic compartments have pNAL-containing N-glycans, yet the CB1 monoclonal binds only to p67 (Brickman and Balber, 1993Go; Kelley et al., 1999Go). Thus, additional p67-specific N-glycan structural feature(s) must contribute to the CB1 epitope. Interestingly the N-terminal gp32 fragment, which contains 4 N-glycosylation sites, is not reactive with either tomato lectin or with CB1 monoclonal (D.L.A. and J.D.B., unpublished). These findings are consistent with the observation that mature gp150 contains some N-glycans that are endoglycosidase H-sensitive — that is, unprocessed (Kelley et al., 1995Go).

Despite the glycan-mediated differences in size of mature p67 in bloodstream and procyclic cells, the sizes of the native proteolytic fragments generated upon arrival in the lysosome are remarkably similar. Comparative analyses of the deglycosylated peptides indicate that cleavage site selection is essentially the same in each lifecycle stage. To reconcile these findings we propose the pNAL modifications of p67 N-glycans in bloodstream cells are removed in the lysosome by endogenous glycosidases and that glycan trimming is concurrent with proteolytic cleavage. Consistent with this hypothesis, tomato lectin blots reveal a heterogeneous smear of steady state p67 polypeptides ranging from 150 to 42 kDa (D.L.A. and J.D.B., unpublished). One potential function of the pNAL modifications may actually be to retard p67 turnover in the more robust lysosome of bloodstream cells, and if so, then turnover would be even more rapid without pNAL epitopes.

Previous work using surface biotinylation in pulse-chase experiments suggested that p67 in bloodstream cells trafficked rapidly through the flagellar pocket en route to the lysosome. Estimates of peak residence in the flagellar pocket were as high as 40% of total radiolabeled p67 (Brickman and Balber, 1994Go), although later experiments suggest that far less newly synthesized p67 is present at any given time [see Fig. 9 in Kelley et al. (Kelley et al., 1995Go)]. We have now used surface biotinylation to assess the steady state level of endogenous p67 polypeptides on the cell surface. Consistently under conditions where cytosolic hsp70 is not accessible, and where transferrin receptor in the flagellar pocket is readily detected, no p67 polypeptides are labeled by external biotinylation. Surface biotinylation is an extremely sensitive assay and this finding indicates that the amount of total p67 in the flagellar pocket is very low, a finding consistent with previous antibody-binding studies on intact trypanosomes (Brickman and Balber, 1993Go). This does not exclude endocytic routing through the flagellar pocket provided that the rate of transit is rapid; however, it now seems likely that trafficking directly from the Golgi may be a significant contributing pathway in bloodstream cells.

p67 targeting signals
The behavior of the p67 truncation reporters in procyclic trypanosomes indicate that lysosomal targeting motif(s) resides in the 24 amino acid cytoplasmic domain. Furthermore, the transmembrane and cytoplasmic domains, but not the transmembrane domain alone, are capable of targeting GFP to the lysosome indicating that cytoplasmic domain is both necessary and likely sufficient for correct targeting in procyclic cells. In addition, overexpressed wildtype p67 leaks to the cell surface suggesting that the machinery for recognition of the specific lysosomal targeting signals is saturable. The two dileucine motifs within the cytoplasmic domain are obvious candidates for such signals (Fig. 1B). In the mammalian endosomal/lysosomal pathway such motifs mediate targeting of membrane proteins, such as mannose 6-phosphate receptor (Johnson and Kornfeld, 1992Go) and LIMP II (Sandoval et al., 1994Go), by serving as ligands for heterotetrameric adapter protein (AP) complexes that in turn mediate assembly of clathrin on budding transport vesicles. Di-leucine motifs can interact with different AP complexes for basolateral sorting (Heilker et al., 1996Go), for endocytosis (Heilker et al., 1996Go) and for alternative post-Golgi pathways (Höning et al., 1998Go). Quantitative and qualitative proof that the p67 di-leucine motifs function in an analogous manner will require other reporters than those used here and this work is currently underway. However, homologues of clathrin and ß-adaptin have been identified in trypanosomes (Morgan et al., 2001Go), and it is a reasonable speculation that these, along with other adaptin homologues, will provide the core machinery for p67 targeting.

The behavior of the p67 reporters in bloodstream trypanosomes is more puzzling. Given the increased endocytic activity of bloodstream cells (Langreth and Balber, 1975Go; Morgan et al., 2001Go), and the fact that p67 can normally traffic through the flagellar pocket, it is not surprising that overexpressed full length p67 does not leak to the plasma membrane. However, the efficient lysosomal delivery of the deletion constructs begs an explanation when they are both quantitatively exported in procyclic cells. One possibility is that an alternative targeting signal is used in bloodstream cells. This signal would have to reside in the lumenal domain to account for lysosomal targeting of p67{Delta}TM and could be a post-translational modification such as the pNAL-containing N-glycans found in bloodstream cells. Nolan et al. have proposed that pNAL on other glycoproteins of the bloodstream endocytic pathway, such as the transferrin receptor, may be a shared epitope specifying internalization via a hypothetical lectin-like receptor in the flagellar pocket (Nolan et al., 1999Go). Such a mechanism could mediate endocytic retrieval of the p67 reporters following delivery to the flagellar pocket. Alternatively, upregulation of a redundant targeting machinery in bloodstream cells could result in recognition of a lumenal p67 epitope, peptide or carbohydrate, that is present in both stages. A second possibility is that lysosomal delivery of the deletion constructs is a nonspecific consequence of the heightened endocytic activity of bloodstream cells. In this scenario, p67{Delta}CD and p67{Delta}TM would enter some post-Golgi compartment at the intersection of the secretory and endocytic pathways, perhaps the flagellar pocket or an internal sorting endosome, at which point they would be carried to the lysosome by `endocytic backflow'. Whatever the alternative mode of targeting, redundancy would benefit the parasite by ensuring that no invariant antigen leaks to the cell surface, where it might elicit potentially lethal host immune responses.

p67 function
p67 has no sequence homology with known mammalian lysosomal proteins, but its overall structure is analogous to LAMP-1 and LAMP-2, both of which have type I membrane topologies with multiple pNAL-containing N-glycans (Hunziker and Geuze, 1996Go). LAMPs are thought to form a continuous protective glycocalyx on the lumenal face of mammalian lysosomes (Granger et al., 1990Go). Surprisingly, gene disruption of mouse LAMP-1 has no effect on basic lysosomal function, although mild effects on brain tissue are seen in live animals (Andrejewski et al., 1999Go). By contrast, genetic deficiencies in LAMP-2 correlate with pathological accumulation of autophagic vacuoles in both humans and mice (Nishino et al., 2000Go; Tanaka et al., 2000Go) suggesting that it will be possible to assign other functions to individual lysosomal membrane proteins by genetic strategies. Although the true function(s) of mammalian LAMPs have not been determined, it may be that p67 plays an analogous role that might also be revealed by genetics. There are too many gene copies (Kelley et al., 1999Go) for a targeted p67 gene disruption approach to work; however, gene silencing by inducible expression of double-stranded RNAi constructs works well in trypanosomes (Ngo et al., 1998Go; Wang et al., 2000Go). Preliminary results with this strategy indicate that ablation of p67 expression leads to rapid cessation of bloodstream trypanosome cell growth followed by cell death (D.L.A. and J.D.B., unpublished). Apparently then, p67 is essential for lysosomal function and in the future trypanosomes should provide a unique model system for studying the role of this general class of membrane proteins.


    Acknowledgments
 
We are indebted to Piet Borst, Conor Caffrey, Gerard Marriott, James McKerrow and David Russell for providing reagents, and to Anant Menon and Derek Nolan for thoughtful discussion and comments. We also thank Amy Andrews for assistance with GFP localization. This work was supported by the National Institutes of Health Grant AI35739 (to J.D.B.). J.D.B. is a Burroughs Wellcome Fund New Investigator in Molecular Parasitology. D.L.A. was supported in part by National Institutes of Health Postdoctoral Fellowship AI10552.


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