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Originally published In Press as doi:10.1074/jbc.M403780200 on July 14, 2004

J. Biol. Chem., Vol. 279, Issue 40, 42106-42113, October 1, 2004
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Endosomes, Glycosomes, and Glycosylphosphatidylinositol Catabolism in Leishmania major*

Zhifeng Zheng{ddagger}, Kimberly D. Butler{ddagger}, Rodney K. Tweten§, and Kojo Mensa-Wilmot{ddagger}

From the {ddagger}Department of Cellular Biology, the University of Georgia, Athens, Georgia 30602 and the §Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

Received for publication, April 5, 2004 , and in revised form, July 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycosylphosphatidylinositols (GPIs) serve as membrane anchors of polysaccharides and proteins in the protozoan parasite Leishmania major. Free GPIs that are not attached to macromolecules are present in L. major as intermediates of protein-GPI and polysaccharide-GPI synthesis or as terminal glycolipids. The importance of the intracellular location of GPIs in vivo for functions of the glycolipids is not appreciated. To examine the roles of intracellular free GPI pools for attachment to polypeptide, a GPI-specific phospholipase C (GPI-PLCp) from Trypanosoma brucei was used to probe trafficking of GPI pools inside L. major. The locations of GPIs were determined, and their catabolism by GPI-PLCp was analyzed with respect to the intracellular location of the enzyme. GPIs accumulated on the endo-lysosomal system, where GPI-PLCp was also detected. A peptide motif [CS][CS]-x(0,2)-G-x(1)-C-x(2,3)-S-x(3)-L formed part of an endosome targeting signal for GPI-PLCp. Mutations of the endosome targeting motif caused GPI-PLCp to associate with glycosomes (peroxisomes). Endosomal GPI-PLCp caused a deficiency of protein-GPI in L. major, whereas glycosomal GPI-PLCp failed to produce the GPI deficiency. We surmise that (i) endo-lysosomal GPIs are important for biogenesis of GPI-anchored proteins in L. major; (ii) sequestration of GPI-PLCp to glycosomes protects free protein-GPIs from cleavage by the phospholipase. In T. brucei, protein-GPIs are concentrated at the endoplasmic reticulum, separated from GPI-PLCp. These observations support a model in which glycosome sequestration of a catabolic GPI-PLCp preserves free protein-GPIs in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycosylphosphatidylinositols (GPIs)1 are present in most eukaryotes (1, 2) in three possible forms: (i) "free" (i.e. unattached to macromolecules); (ii) linked to proteins (e.g. gp63 of Leishmania spp. and Thy-1 of vertebrates); or (iii) attached to polysaccharides (e.g. lipophosphoglycan of Leishmania).

In the protozoan parasite Leishmania major, free GPIs are detected in two general classes, namely protein-GPIs and polysaccharide-GPIs. Protein-GPIs contain the core glycolipid Man{alpha}1-6Man{alpha}1-4GlcNH2-PI. They include intermediates of GPI biosynthesis (e.g. GlcNH2-PI and Man{alpha}1-6Man{alpha}1-4GlcNH2-PI) and the complete protein anchor ethanolaminephospho-Man{alpha}1-2Man{alpha}1-6Man{alpha}1-4GlcNH2-PI (3, 4). Polysaccharide-GPIs contain Man{alpha}1-3Man{alpha}1-4GlcNH2-PI (4).

Free protein-GPIs are synthesized in the secretory system, beginning on the cytoplasmic side of the endoplasmic reticulum (ER) (5, 6) and are detectable on the plasma membrane of vertebrates and trypanosomatids (7-11). The exocytic system is continuous yet highly compartmentalized; it includes the ER, Golgi, secretory vesicles, and in highly polarized cells (e.g. trypanosomatids) parts of the endosomal system where the secretory and endocytic pathways converge (12, 13).

In L. major, the importance of intracellular pools of GPIs has not been studied in the context of biological functions. For example, it is held that GPIs are synthesized within the ER, yet there is no direct evidence that GPIs are added to proteins in ER. Only in the yeast Saccharomyces cerevisiae has it been demonstrated conclusively that when anterograde vesicle traffic is blocked GPIs are added to protein in the ER (14).

Because GPIs are present on the plasma membrane of Leishmania, it stands to reason that many areas of the secretory pathway might contain the glycolipids (8). Unanswered questions in the field include the following: Do GPIs accumulate to different extents in different organelles of the endo/exocytic pathway of Leishmania? Are all GPIs in the secretory pathway equally competent for addition to proteins? Are GPIs added to protein in the ER? How is the biosynthesis/storage of GPIs coordinated with catabolic enzymes that might cleave the glycolipids? These questions deserve to be addressed for Leishmania and other eukaryotes because there are clear differences between vertebrate cells, for example, and S. cerevisiae in the molecular machinery and pathways for endo/exocytosis (15-17).

Trypanosoma brucei expresses a GPI-specific phospholipase C (GPI-PLCp) (18-20). Heterologous (stable) expression of GPI-PLCp in L. major confers a GPI-negative phenotype on the cells by depleting intermediates of protein-GPI biosynthesis (21). The shortage of prefabricated GPIs for anchoring polypeptides to membranes leads to constitutive secretion of major cell surface polypeptides (e.g. gp63) that should have been GPI-anchored. These proteins enter the ER because of their N-terminal signal peptide but lack signals for retention in the cell (21, 22). Polysaccharide-GPIs are not cleaved in vivo by GPIPLCp in L. major (21).

Peroxisomes belong to the microbody group of organelles (for review, see Ref. 23). Important for both catabolic (e.g. {beta}-oxidation of fatty acids, decomposition of hydrogen peroxide) and biosynthetic reactions (e.g. ether-lipid and cholesterol), peroxisomes in different eukaryotes may contain specialized pathways (e.g. glyoxylate cycle in plants and purine synthesis in mammals (for review, see Ref. 23)). The trypanosomatids (e.g. Leishmania spp. and T. brucei) are deeply diverged eukaryotes (24, 25). In these organisms, peroxisomes, in addition to being the organelle in which ether-lipid synthesis and {beta}-oxidation of fatty acids take place, harbor the first seven enzymes of the glycolysis pathway (for review, see Ref. 26). Consequently, peroxisomes are termed "glycosomes." Mistargeting of glycolytic enzymes to the cytosol is lethal in the bloodstream form of T. brucei (27-29), indicating that glycosomes are an essential organelle in the parasite.

We used the GPI-binding protein {alpha}-toxin from Clostridium septicum (30, 31) to identify sites of free protein-GPI accumulation in L. major.2 Concurrently, GPI-PLCp was used as a "catabolic probe" of free protein-GPIs in Leishmania. Both GPIPLCp and protein-GPIs were detected on the endo-lysosomal system of L. major. However, several cysteine mutations in GPI-PLCp caused the enzyme to localize to glycosomes. Glycosomal GPI-PLCp did not cause a protein-GPI deficiency in L. major. Thus, endo-lysosomal GPIs are important for GPI addition to protein. Further, targeting of GPI-PLCp to glycosomes sequesters the enzyme from free protein-GPIs in vivo. Data obtained in T. brucei where GPI-PLCp is endogenous are consistent with the "GPI sequestration" hypothesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—A GPI-phospholipase C plasmid pUTK-GPIPLC was obtained by insertion of a GPI-PLC coding region (19, 32) and a translation enhancing 5'-untranslated region (33) into a Leishmania expression plasmid pXUTE-KanaR (pUTK) (34). The GPI-PLC insert was generated by PCR using a forward primer KCR4 (5'-TAAGGATCCTTAACACAGGAGGCAGCTAatgtttggtggtgta-3') and the reverse primer KCR5 (5'-TATGTGGATCCTTAtgaccttgcggtttggt-3'). KCR4 has a BamHI site (underlined) followed by a stop codon (italicized), an AUG-proximal region (e.g. lacZ-CTA (italicized), and finally a sequence encoding the first 4 amino acids of GPI-PLCp (in lowercase lettering) (33, 35). The reverse primer KCR5 contains a BamHI site (underlined), a stop codon (bold italicized), and the last 7 amino acids of GPI-PLCp (lowercase) (19, 32). The amplification product was digested with BamHI and ligated into a BglII site of pUTK (34).

Site-directed mutagenesis was performed on pUTK-GPIPLC using a GeneEditor kit (Promega; Madison, WI). Primer sequences for each set of cysteine mutations were as follows (mutated nucleotides are in bold), C24A, 5'-TTGACCAATCGCTTTCTTCTCAATGG-3'; C80A, 5'-AGAAAGATTTTGCGCACGTCC CCA-3'; C184A, 5'-CTGCCAGAGGTTCGC- AAGTGGTGT-3'; C269S/C270S, 5'-GCGGTGGCAGCGTCTTCTGGCGCGTGTCCC-3'; C269S/C273S, 5'-GCGGTGGCAGCGTCTTGTGGCGCGTCTCCCGGTTCACAT-3'; C270S/C273S, 5'-GCAGCGTGTTCT- GGCGCGTCTCCCGGTTCACAT-3'; C269S/C270S/C273S, 5'-GCGGTGGCAGCGTCTTCTGGCGCGTCTCCCGGTTCACAT-3'; C332A, 5'-GAAGGCACTGCGACTGTTAAGGGA-3'; and C347A, 5'-GTTGCATTA-GCGGTTCATTTAAACA CC-3'. All mutations were verified by DNA sequencing (Integrated Biotechnology Laboratories, University of Georgia, Athens).

Leishmania Transfection—L. major promastigote strain LT252-CC1 (36) was cultivated at 27 °C in M199 medium supplemented with 10% fetal bovine serum. Cells (1 x 107/ml) were harvested by centrifugation at 2,000 x g for 5 min. Cell pellets were washed once with ice-cold electroporation buffer (21 mM HEPES, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2PO4, 6 mM glucose) and electroporated with the following conditions; 475 V, 800 microfarads, 13 ohms, one-pulse (BTX ECM-600 apparatus) (37). The cells were incubated for 8-12 h at 27 °C before the addition of 30 µg/ml G418 to select stable transfectants. When specified, cells were grown in medium containing 200 µg/ml G418 for 21-28 days before use (see appropriate figure legends).

GPI-PLC Enzyme Assay—Leishmania (1 x 107 cells/ml) growing in medium containing 200 µg/ml G418 were harvested, washed in PBS and lysed in 1 ml of hypotonic buffer (10 mM Tris-HCl, pH 8.0, 2 mM EDTA) containing a protease inhibitor mixture (2.1 µM leupeptin, 0.1 mM N-tosyl-L-lysine chloromethyl ketone, 0.4 unit aprotinin) (38). The cells were incubated on ice for 20 min and then centrifuged for 20 min (14,000 x g, 4 °C). The pellet was resuspended in 500 µl of GPI-PLC assay buffer AB (1.0% Nonidet P-40, 5 mM EDTA, 50 mM Tris-HCl, pH 8.0) and incubated on ice for 20 min. After centrifugation (14,000 x g, 20 min, 4 °C), portions of the supernatant were added to 15 µl of GPI-PLC assay buffer containing 2 µg of [3H]myristate-labeled membrane form variant surface glycoprotein ([3H]mfVSG) (38). The reaction mixture was incubated at 37 °C for 20 min and terminated by the addition of 500 µl of water-saturated butanol. The amount of [3H]dimyristoylglycerol released from [3H]mfVSG was quantified by scintillation counting of 400 µl of the organic phase. Lysates were titrated to ensure that the amount of enzyme measured was within the linear range of the assay (i.e. 0.1-1.0 unit) (38). Total protein concentration of cell lysates was determined with a bicinchoninic acid assay (Pierce).

Western Blotting—For detection of gp63, parasites were lysed in 1 ml of hypotonic buffer (10 mM Tris-HCl, pH 8.0, 2 mM EDTA) containing protease inhibitor mixture and 5 mM p-chloromercuriphenyl sulfonate followed by incubation on ice for 15 min. The lysate was centrifuged (14,000 x g, 4 °C, 20 min), and the pellet was resuspended in high salt buffer (10 mM Tris-HCl, pH 8.0, 2 mM EDTA, 500 mM NaCl) containing protease inhibitor mixture and 5 mM p-chloromercuriphenyl sulfonate. After centrifugation (14,000 x g, 4 °C, 10 min) and washing with hypotonic buffer containing protease inhibitor mixture and 5 mM p-chloromercuriphenyl sulfonate, the pellet was resuspended in 80 µl of 2.5x SDS-PAGE sample buffer. Solubilized proteins were separated by SDS-PAGE (14% minigel) and transferred to Immobilon P membrane. Western blotting of the membrane with anti-gp63 (1:2,000) (39) was carried out as described previously (40).

To monitor glycoproteins to which concanavalin A (ConA) bound, the Immobilon P membrane (see preceding paragraph) was blocked in 5% nonfat milk (Carnation) in PBS and treated with 5 µg/ml ConA for 1 h at 27 °C. The membrane was washed with PBS followed by incubation with anti-ConA antibody (Sigma) (1:6,000 dilution) for 1 h at room temperature. After washing with PBS, the membrane was incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Roche Applied Science) (1:1,000 dilution) for 1 h and then processed for antigen detection with 5-bromo-4-chloro-3-indolyl phosphate p-toluidine/nitro blue tetrazolium chloride (41).

Organelle Detection in Live Cells—Endo-lysosomal compartments were identified after uptake of three fluorescent markers: dextran-Texas Red, FM4-64, and LysoTracker Red. For endocytosis of dextran-Texas Red, Leishmania at a density of 1 x 107 cells/ml were gently pelleted (2,000 x g, 2 min) and resuspended in 500 µl of M199 medium containing 500 µg/ml dextran-Texas Red (Molecular Probes, Inc.) at 27 °C for 10 or 30 min. Cells were also stained (in 500 µl of M199 medium) with 10 µM FM4-64 (Molecular Probes, Inc.) froma4mM stock in dimethyl sulfoxide). Acidic organelles (endosomes and lysosomes) were stained with 100 nM LysoTracker Red (Molecular Probes, Inc.) at 27 °C for 5 min-1 h.

Golgi complex was labeled with BODIPY-TR ceramide (Molecular Probes, Inc.). Leishmania were incubated with 5 µM BODIPY-TR ceramide bound to defatted bovine serum albumin for 90 min (1 h at 4 °C, and 30 min at 27 °C) (42).

To detect protein-GPIs, {alpha}-toxin (2.5 µg/ml in PBS) was added to fixed/permeabilized or nonpermeabilized cells (30 min at 27 °C) followed by ConA-Alexa Fluor 594 (5 µg/ml) (Molecular Probes, Inc.) (30 min at 27 °C).2 {alpha}-Toxin was detected with anti-{alpha}-toxin antibody (1:1,500) (30) and Alexa Fluor 488-conjugated anti-rabbit secondary antibody.

A C-terminal fusion of green fluorescent protein (GFP) to GPI-PLCp (i.e. GPIPLCp-GFP) was visualized in live L. major as described previously (43). DNA was stained with DAPI (10 µM in CitiFluor solution (CitiFluor Ltd.)).

Immunofluorescence Assays—Log phase cells (1 x 107/ml) were fixed in 2% paraformaldehyde (8 min, on ice), washed in PBS, and adhered to poly-L-lysine-coated coverslips. The cells were permeabilized with 0.25% (w/v) Triton X-100 (200 µl/coverslip) for 5 min at 4 °C. Nonspecific sites were blocked with 1% bovine serum albumin and PBS for 1 h at room temperature. For studies with T. brucei bloodstream, cells were fixed as described but permeabilized with methanol (5 min, -20 °C) before processing as described below.

To detect GPI-PLCp, anti-GPIPLC (anti-peptide) antibody RC300 (1:1,200 dilution in blocking solution; 200 µl total volume) or monoclonal 2A6-6 (1:1,500 dilution in blocking solution; 200 µl total volume) (32) was added for 1 h at room temperature. Cells were washed once with PBS, twice with high salt buffer (PBS plus 500 mM NaCl), and twice with PBS. The cells were then immersed in 200 µl of a 1:1,000 dilution of Alexa Fluor 488-conjugated goat anti-rabbit IgG or a 1:2,000 dilution of Alexa Fluor 594-conjugated goat anti-mouse IgG for 1 h at 4 °C.

The ER was visualized by detection of BiP with anti-BiP antibody (1:2,000 dilution) (44). Glycosomes were detected with antibody against hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (45) (a 1:1,000 dilution).2 Intracellular GPIs were detected with 2.5 µg/ml {alpha}-toxin in combination with 1:1,500 dilution of anti-{alpha}-toxin antiserum (31). The cell nucleus and kinetoplast (mitochondrial DNA) were stained with 10 µM DAPI in CitiFluor.

Labeled cells were viewed with a fluorescence microscope (Leica DMIRBE). Images were captured using an interline chip-cooled CCD camera (Orca 9545: Hamamatsu) and processed with OpenLab 3.1.2 software (Improvision, Inc.).

In both organelle detection and immunofluoresence assays, more than 100 cells were visualized microscopically. Data presented in each figure are representative of those found in at least 90% of the population, unless otherwise noted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Exploration of GPI Utilization in L. major—To investigate functions of intracellular pools of free GPIs in L. major, a "probe" for GPIs was needed in living cells. GPI-PLCp from T. brucei was highly suitable as the in vivo probe of GPIs because heterologous expression of the enzyme in L. major causes a deficiency of (free) protein-GPI without affecting polysaccharide-GPI metabolism (21, 46). L. major expressing GPI-PLCp or cysteine mutants of the enzyme were analyzed for two reasons. First, most Cys mutants of GPI-PLCp (expressed in Escherichia coli) retain enzyme activity (47, 48). Second, in some biological systems Cys mutations can alter intracellular location of polypeptides (49-52). Therefore, we reasoned that Cys mutants of GPI-PLCp, compared with unmutated enzyme, could have different intracellular locations that might lead to divergent effects of the enzyme on GPIs in L. major.

For our work, mutations of Cys to alanine (Ala) or serine (Ser) in GPI-PLCp were constructed. It was important to test whether the altered proteins retained enzyme activity in L. major. For this purpose, in vitro experiments were performed; lysates of L. major transfectants expressing GPI-PLCp or Cys mutants of the enzyme (e.g. C80A) were used to cleave exogenous [3H]myristate-labeled VSG (38) (Fig. 1A). Cys mutants of GPI-PLCp exhibited enzyme activity in the range detected for the unmutated protein (Fig. 1A). No GPI-PLCp activity was detected in L. major transfectants expressing pUTK (34), the expression vector without a gene for GPI-PLCp. A Q81L mutant of GPI-PLCp was devoid of enzyme activity (Fig. 1A). These observations are consistent with data from earlier studies of enzyme activity in E. coli (34, 47, 48).



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  		FIG. 1.
A, activity of GPI-PLCp cysteine mutants in lysates of L. major. Detergent extract from L. major expressing GPI-PLC was assayed for enzyme activity and corrected for total protein. 100% activity corresponded to 652 units/mg activity. B, effect of GPI-PLCp on GPI-anchoring of Gp63p in L. major. Parasites were lysed hypotonically in the presence of p-chloromercuriphenyl sulfonate, and the membrane-bound proteins were separated by SDS-PAGE. Proteins were transferred to Immobilon P membrane and detected with anti-gp63 polyclonal antibody or by ConA immunoblotting.

 
Heterologous expression of GPI-PLCp in L. major produces a GPI deficiency that is marked by accelerated secretion of gp63, the major GPI-anchored protein (21, 53). Loss of gp63 from the cells was used as a "read-out" of increased catabolism of (free) protein-GPIs in vivo. From Western blot analysis (Fig. 1B), gp63 was reduced in cells expressing GPI-PLCp (Fig. 1B, compare lane 1 with lane 2). Deficiency of cell-associated gp63 also occurred in L. major expressing C184A and C347A mutants of GPI-PLCp (termed class II Cys mutants) (Fig. 1B, lanes 4 and 9). In contrast, L. major transfected with C80A, C269S/C270S, C269S/C273S, C270S/C273S, and C269S/C270S/C273S mutants of GPI-PLCp (termed class I Cys mutants) retained gp63 (Fig. 1B compare lanes 3, 5, 6, 7, and 8 with lanes 4 and 9). This last result indicated that L. major containing the latter (class I) Cys mutants of GPI-PLCp were no longer GPI-deficient. Likewise, L. major expressing Q81L, an enzymatically inactive mutant of GPI-PLCp (48), contained a normal amount of gp63 (Fig. 1B compare lane 10 with lane 2).

We infer that specific Cys mutants of GPI-PLCp (i.e. C80A, C269S/C270S, C269S/C273S, C270S/C273S, and C269S/C270S/C273S) suppress GPI-PLCp catabolism of GPIs in vivo. Further, GPI-PLCp enzyme activity is required to produce a GPI deficiency in L. major because cells expressing a Q81L mutant of GPI-PLCp were not GPI-deficient.

Altered Intracellular Location of Class I Cys Mutants of GPIPLCp—We were interested in learning why some but not all Cys mutants of GPI-PLCp failed to produce GPI-deficient L. major (Fig. 1B). Because all of the proteins cleaved GPIs in vitro (Fig. 1A), an explanation that some Cys mutants were enzymatically inactivated was ruled out, in favor of an alternative theory that Cys mutations in GPI-PLCp altered intracellular targeting of the enzyme. To set the stage for testing this second hypothesis, we first determined the intracellular location of unmutated GPI-PLCp in L. major.

In immunofluorescence analysis, GPI-PLCp was detected at the anterior end of Leishmania, frequently positioned between the nucleus and the kinetoplast (mitochondrial DNA). A smaller amount of the protein was detected posterior to the cell nucleus (Fig. 2, C and D). In control experiments, cells containing the expression vector (pUTK) alone had no fluorescence (data not shown). These results demonstrate that GPI-PLCp associates with an organelle (or structure) in the anterior region of L. major.



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  		FIG. 2.
GPI-PLCp is not detected at the Golgi or ER. A-D, Leishmania transfected with unmutated GPI-PLCp was fixed, permeabilized, and incubated with anti-GPIPLCp monoclonal antibody, followed by Alexa Fluor 594 goat anti-mouse-conjugated IgG (red). E-H, L. major expressing GPI-PLCp was fixed, permeabilized, and incubated with anti-GPIPLCp and anti-BiP antibodies. Anti-GPIPLC was detected using Alexa Fluor 594 goat anti-mouse IgG conjugate (G, red). Anti-BiP was detected by an Alexa Fluor 488 goat-conjugated anti-rabbit IgG (F, green). I-L, cells expressing GPIPLC-GFP were incubated with the Golgi marker, BODIPY-TR ceramide. GPIPLC-GFP was detected by standard protocols (J, green); BODIPY-TR ceramide (K, red) and a merge of images from J and K are shown on L. Blue, DAPI staining of DNA; n, nucleus; k, kinetoplast (mitochondrial DNA). More than 100 cells were visualized microscopically. Data presented in each panel are representative of at least 90% of the population.

 
Protein-GPI biosynthesis begins on the cytoplasmic side of membranes of the secretory system, which includes the ER (5, 6). One hypothesis to explain the GPI deficiency in Leishmania expressing GPI-PLCp is that the enzyme binds to the ER where it digests cytoplasmically oriented (free) protein-GPIs (21). (Lacking an N-terminal signal sequence (19, 32), GPI-PLCp cannot enter the secretory system.) To test this hypothesis, Leishmania expressing GPI-PLCp were double labeled with antibodies against BiP, an ER marker (44) and GPI-PLCp, and observed by fluorescence microscopy. Some BiP was found at the periphery of the cell nucleus, but the protein was concentrated in a region anterior to the kinetoplast (Fig. 2F). In contrast, GPI-PLCp was located largely between the nucleus and the kinetoplast (Fig. 2G). Therefore, the location of GPIPLCp is distinct from the ER. A small fraction of GPI-PLCp appeared to overlap with a minor proportion of BiP in the region between the nucleus and the kinetoplast. Nonetheless, the general staining patterns of GPI-PLCp and BiP are different, justifying a preliminary conclusion that GPI-PLCp is not an ER protein. These data alone, however, cannot exclude the possibility that GPI-PLCp is targeted to a subdomain of the ER.

GPI-PLCp did not localize to the Golgi complex. This fact was demonstrated by labeling L. major expressing a GPIPLC-GFP chimera with the Golgi marker BODIPY-TR ceramide (42). The fluorescence from GPIPLC-GFP (Fig. 2J) was not coincident with the signal from BODIPY-TR ceramide (Fig. 2, K and L). (In control experiments, the GFP tag did not change the location of GPI-PLCp; compare Fig. 2D with Fig. 2J or 2G.)

GPI-PLCp Associates with the Endo-lysosomal System of L. major—We investigated whether (or not) GPI-PLCp could be targeted to the endo-lysosomal system because portions of those organelles are found between the nucleus and kinetoplast (12, 13, 43), similar to GPI-PLCp (Figs. 2 and 3; see also Fig. 5). To test this hypothesis, endosomes in L. major expressing GPIPLC-GFP were detected with the styryl dye FM4-64 (43, 54). After endocytosis of FM4-64 for 30 min, the dye and GPIPLC-GFP were visualized in live cells. FM4-64 encompassed the region where GPI-PLCp was found (Fig. 3, B-D), indicating that GPI-PLCp associated with the endo-lysosomal system of Leishmania. To distinguish between early and late endosomal compartments, parasites were allowed to endocytose fluorescent dextran-Texas Red for 1, 3, 5, 30, and 45 min. Dextran-Texas Red was not detected between the kinetoplast and the nucleus before 10 min (data not shown). After 30 min, the dye colocalized with GPIPLC-GFP (Fig. 3, F-H). We conclude that GPI-PLCp associates with late endosomes (and possibly the lysosomal system) but not early endosomes.



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  		FIG. 3.
GPI-PLCp associates with the endo-lysosomal system of L. major. A-D, uptake of FM4-64 into endosomes. Leishmania expressing GPIPLC-GFP was exposed to 10 µM FM4-64 for 30 min at 27 °C. Green fluorescence from GPIPLC-GFP (B, green), FM4-64-labeled structures (C, red), and overlay of images (D) are shown. E-H, L. major was labeled with dextran-Texas Red for 30 min at 27 °C. A GFP-labeled structure (F, green), dextran (G, red), and the digital overlay image (H) of both are presented in the far right panel. I-L, cells were exposed to 100 nM LysoTracker Red for 30 min at 27 °C. Green fluorescence of GPIPLC-GFP (J), LysoTracker Red (K, red), and a merged image (L) are shown. Data presented in the panels are representative of at least 90% of more than 100 cells examined.

 



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  		FIG. 5.
Some cysteine mutants of GPI-PLCp associate with the endo-lysosomal system. Leishmania transfectants expressing pUTKGPIPLC-C184A (A-D) or pUTK-GPIPLC-C347A (E-H) were incubated with 10 µM FM4-64 for 30 min at 27 °C. Cells were fixed, permeabilized, and labeled with rabbit anti-GPIPLC RC300 antibody followed by detection with Alexa Fluor 488 goat ant-rabbit IgG-conjugated secondary antibody (green). FM4-64-labeled structures (C and G, red) and overlaid images (D and H) are shown. Data presented in each panel are representative of at least 90% of more than 100 cells examined.

 
Further evidence that GPI-PLCp was present on the endolysosomal system was obtained by staining L. major with LysoTracker Red, a dye that accumulates in organelles with low pH (12, 43). In double labeling studies, the green fluorescence from GPIPLC-GFP was coincident predominantly with zones detected with LysoTracker Red (Fig. 3L). Similar results were obtained in experiments using a nontagged form of GPI-PLCp (data not presented).

From these observations, we conclude that GPI-PLCp is targeted specifically to the endo-lysosomal system in L. major. GPI-PLCp is highly likely to bind to the cytoplasmic side of endosomes because it lacks an N-terminal ER signal peptide for entry into the secretory pathway (19, 32).

Glycosomal Targeting of Class I Cysteine Mutants of GPIPLCp—Knowing the location of GPI-PLCp in Leishmania (Figs. 2 and 3) it was possible to test whether or not Cys mutations in the polypeptide redirected the enzyme to a different site. Following protocols used to determine the location of unmutated GPI-PLCp, the C184A and C347A mutants of GPIPLCp (both of which are class II Cys mutants, defined as enzymes that produced a protein-GPI deficiency in L. major) were found in the anterior region of L. major, similar to unmutated GPI-PLCp (Fig. 4, A and B). The Q81L mutant was also detected in the anterior region (Fig. 4C).



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  		FIG. 4.
Localization of cysteine mutants of GPI-PLCp in L. major. Leishmania transfected with cysteine mutants of GPI-PLC were fixed and permeabilized. Anti-GPIPLC monoclonal antibody was added followed by detection with Alexa Fluor 594 goat anti-mouseconjugated IgG (red). DNA was stained with DAPI (blue). In each pair of images, the phase-contrast image is on the left, and a merged fluorescence image is on the right; n, nucleus; k, kinetoplast. More than 100 cells were visualized microscopically. Data presented in each panel are representative of at least 90% of the population.

 
Class I Cys mutants of GPI-PLCp (defined as those proteins that failed to produce GPI-deficient L. major) included C80A, C269S/C270S, C269S/C273S, C270S/C273S, and C269S/C270S/C273S. Those proteins were associated with punctate structures throughout the cell (Fig. 4, D-H). Apparent displacement of GPI-PLCp from the anterior region of Leishmania in the class I mutants correlates with reversal of the GPI deficiency normally associated with GPI-PLCp expression in vivo (Fig. 1B).

The organelle that GPI-PLCp bound in L. major was identified in double labeling experiments with anti-GPIPLCp (antibody) and one of four markers of subcellular organelles:FM4-64, dextran-Texas Red, anti-HGPRT, and anti-aldolase. (HGPRT and aldolase are marker proteins for peroxisomes (glycosomes) of Leishmania (45), whereas FM4-64 and dextran-Texas Red are taken up into endosomes and lysosomes (12, 55).) C184A and C347A mutants of GPI-PLCp colocalized with endocytosed FM4-64 (Fig. 5). Similar data were obtained with dextran-Texas Red (not presented). These observations demonstrate that class II Cys mutants of GPI-PLCp, which retain the ability to cause a GPI deficiency, associate with the endolysosomal system. However, the class I mutants of GPI-PLCp (i.e. C80A and C269S/C270S/C273S) did not colocalize with the endosomal system (data not presented).

Class I Cys mutants of GPI-PLCp were targeted to glycosomes because they colocalized predominantly with HGPRT (Fig. 6, A-H) (and aldolase, not presented). Control (unmutated) GPI-PLCp did not colocalize with HGPRT (Fig. 6, I-L). Together these results demonstrate that specific Cys mutants of GPI-PLCp associate with glycosomes.



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  		FIG. 6.
Peroxisomal targeting of a group of cysteine mutants of GPI-PLCp. Leishmania transfected with cysteine mutants of GPIPLCp (C80A (A-D) or C269S/C270S/C273S (E-H) and unmutated GPIPLCp (I-L) were fixed, permeabilized, and double stained with rabbit anti-HGPRT (C, G, and K) and anti-GPIPLC monoclonal (B, F, and J) antibodies. Rabbit anti-HGPRT and mouse anti-GPIPLC were detected with Alexa Fluor 488 goat anti-rabbit IgG conjugate (green) and Alexa Fluor 594 goat anti-mouse IgG conjugate secondary antibody (red), respectively. Blue staining, DAPI for nucleus (n) and kinetoplast (k). More than 100 cells were examined in each experiment. Data presented in each panel are representative of at least 90% of the cells examined.

 
Endosomal GPIs: Colocalization with GPI-PLCp Causes a GPI Deficiency—Stable expression of GPI-PLCp in Leishmania causes a protein-GPI deficiency (21, 46). Interestingly, enzyme activity of GPI-PLCp (Fig. 1B) and the presence of the enzyme on the endo-lysosomal system (Figs. 3 and 4) are necessary to produce GPI-deficient cells. Based on these observations, we envisioned that both GPIs and GPI-PLCp might be detected concurrently on endosomes if the enzyme did not cleave all of the glycolipids. To test this theory, we attempted to find (free) protein-GPIs and GPI-PLCp in L. major expressing either unmutated or the enzymatically inactive Q81L mutant of GPIPLCp. Similar studies were performed with a C269S/C270S/C273S mutant of GPI-PLCp as a control.

Intracellular GPIs detected with the GPI-binding protein {alpha}-toxin were found on the endo-lysosomal system.2 Those GPIs colocalized with unmutated GPI-PLCp (Fig. 7, B-D). Unfortunately, because unmutated GPI-PLCp cleaves GPIs, the intensity of the {alpha}-toxin fluorescence was routinely less than that found in cells that did not contain the phospholipase. To preserve the {alpha}-toxin fluorescence signal, we performed similar studies with the Q81L mutant of GPI-PLCp, which does not cleave GPIs (48). In L. major containing the Q81L mutant, colocalization of GPI and GPI-PLCp was clear (Fig. 7, I-L) compared with cells containing unmutated enzyme (Fig. 7, B-D). These data indicate that GPI-PLCp binds to (free) protein-GPIs on endosomes. In contrast, the vast majority of the C269S/C270S/C273S mutant (Fig. 7F) failed to colocalize with {alpha}-toxin (Fig. 7, G and H). Thus, the inability of C269S/C270S/C273S GPI-PLCp (and other class I Cys mutants) to produce GPI-deficient Leishmania correlates with failure of the bulk of the proteins to bind endosomes.



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  		FIG. 7.
Intracellular GPIs colocalize with GPI-PLCp. Leishmania harboring pUTK-GPIPLCp (A-D), pUTK-GPIPLC-C269S/C270S/C273S (E-H), or pUTK-GPIPLC-Q81L (I-L) were cultured in medium containing 30 µg/ml G418. Cells were fixed with paraformaldehyde and permeabilized with Triton X-100. The locations of GPI-PLCp and GPIs were detected with mouse anti-GPIPLC 2A6.6 (B, F, and J, red) and {alpha}-toxin/rabbit anti-{alpha}-toxin (C, G, and K, green) antibodies. The positions of the nucleus and kinetoplast are shown by DAPI staining (blue). Data presented in each panel are representative of at least 90% of more than 100 cells examined.

 
T. brucei: GPIs Accrue at the ER and Are Sequestered from GPI-PLCp—The experiments in Leishmania led to the conclusion that free protein-GPIs can be protected from cleavage by GPI-PLCp if the enzyme is targeted to glycosomes in vivo (Figs. 1B and 6). In T. brucei where GPI-PLCp is endogenous, the enzyme does not cause a GPI deficiency that results in constitutive secretion of the major GPI-anchored protein VSG (18, 56, 57). Therefore, we hypothesized that free protein-GPIs may be separated from GPI-PLCp in T. brucei. To test this theory, we examined the location of both intracellular GPIs and GPI-PLCp (Fig. 8). Intracellular GPIs were detected with the GPI-binding protein {alpha}-toxin,2 whereas endosomes were detected after endocytosis of FM4-64 (38, 55, 59).



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  		FIG. 8.
T. brucei: sequestration of GPI-PLCp from (free) GPI. A-D, intracellular GPIs localize to a subregion of the ER. Fixed, permeabilized bloodstream form cells were incubated with 2.5 µg/ml {alpha}-toxin for 30 min followed by anti-{alpha}-toxin antibody. Biotinylated antibody to the ER membrane protein Sec61p was added; it was detected with streptavidin-Alexa Fluor 594. The anti-{alpha}-toxin antibody was localized with Alexa Fluor 488 goat anti-rabbit IgG. E-H, intracellular GPIs do not accumulate on endosomes of T. brucei. Bloodstream form CA427 were allowed to endocytose 10 µM FM4-64 for 10 min at 37 °C, fixed, and permeabilized (0.05% CHAPS). The cells were incubated with 2.5 µg/ml {alpha}-toxin for 30 min and detected by anti-{alpha}-toxin antibody and Alexa Fluor 488 goat anti-rabbit IgG antibody. I-L, GPI-PLCp is a glycosome protein in T. brucei. Bloodstream form cells were fixed, permeabilized, and incubated with monoclonal 2A6.6 anti-GPIPLCp antibody and rabbit anti-aldolase antibody. The two antibodies were detected with Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG. M-P, endogenous GPI-PLCp is sequestered from intracellular GPIs in T. brucei. Fixed, permeabilized bloodstream form cells were incubated with 2.5 µg/ml {alpha}-toxin for 30 min followed by anti-{alpha}-toxin antibody and monoclonal 2A6.6 anti-GPIPLC antibody. Antibodies were detected with Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG. The cell nucleus (n) and the kinetoplast (k) were detected with DAPI staining (blue). {alpha}-Toxin antibody labeling (B, F, and N), TbSec61p-biotin antibody-labeled sites (C), aldolase antibody-labeled structure (J), GPI-PLCp antibody-labeled structure (K and O), and the electronically overlaid images (D, H, L, and P) are presented. Data presented in each panel are representative of at least 90% of more than 100 cells examined.

 
GPIs were detected in a subregion (i.e. the posterior) of the ER, which was identified with the marker protein TbSec61p (Fig. 8, A-D). (Sec61p forms the protein import pore on the ER of eukaryotes (60).) GPIs were not concentrated on the endolysosomal system of T. brucei (Fig. 8, E-H). GPI-PLCp was associated with glycosomes, which were detected with the marker protein aldolase (61) (Fig. 8, I-L). Consequently, GPIs in T. brucei did not colocalize with GPI-PLCp (Fig. 8, M-P).

In conclusion, the bulk of free protein-GPIs in T. brucei were detected at the ER. These GPIs are sequestered from GPI-PLCp, which is glycosomal in T. brucei (Fig. 8L). Separation of enzyme from substrate is likely to help preserve the protein-GPIs that are needed for attachment to proteins (e.g. transferrin receptor and VSG) in T. brucei (for review, see Refs. 2 and 62).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Endosomal GPIs are Important for Biogenesis of GPI-anchored Proteins in L. major—In this study, we have found two conditions that facilitate catabolism of (free) GPIs in L. major (outlined in Fig. 9). First, enzyme activity of GPI-PLCp is necessary because a Q81L mutant of the protein which cannot cleave GPIs (48) fails to produce GPI-deficient L. major (Fig. 1). Second, targeting of GPI-PLCp to intracellular GPIs is important, as expected. In L. major, free protein-GPIs accumulate at endosomes (Fig. 3).2 Consequently, cells become GPI-deficient only if GPI-PLCp associates with the endo-lysosomal system (Figs. 3 and 5). When Cys mutants of GPI-PLCp are targeted to glycosomes, the GPI deficiency is averted (Figs. 6 and 9). These observations highlight the importance of endosomal GPI in biogenesis of GPI-anchored proteins in L. major: deficiency of protein-GPIs is associated with loss of cell-associated gp63 (Fig. 1B) (21).



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  		FIG. 9.
A sequestration model for regulation of GPI-PLCp in Leishmania. Protein-GPIs are associated mainly with the endo-lysosomal system. Colocalization of enzymatically active GPI-PLCp and protein-GPIs on endosomes leads to a GPI deficiency, arising from cleavage of protein-GPIs by the phospholipase C. Peroxisomal targeting of GPI-PLCp separates the enzyme from protein-GPIs present on the endo-lysosomal system. A GPI-negative phenotype can be obviated by sequestration of GPI-PLCp from its potential intracellular substrate. Although directed to endosomes, a catalytically inactive mutant (Q81L) of GPI-PLCp does not produce a protein-GPI deficiency in Leishmania because it does not cleave GPIs efficiently.

 
In T. brucei where GPI-PLCp is endogenous (18, 20, 57), each cell contains ~107 molecules of VSG, a GPI-anchored protein (63). How does T. brucei retain sufficient GPIs needed for anchoring VSG to the plasma membrane when GPI-PLCp is present in the same cell? The data obtained from the class I Cys mutants of GPI-PLCp in our studies in L. major suggest a plausible model: separation of GPI-PLCp from areas of the cell where (free) GPIs accumulate may help prevent cleavage of GPIs. This so-called GPI-PLCp sequestration hypothesis was evaluated in T. brucei. There, we discovered that GPIs accumulate at the ER, whereas GPI-PLCp is a glycosome protein (Fig. 8). These data are consistent with a proposal that sequestration of GPI-PLCp to glycosomes contributes to prevention of excessive cleavage of GPIs in T. brucei (as first observed in L. major). (Signals that target GPI-PLCp to glycosomes in T. brucei and L. major are under investigation because the enzyme lacks peroxisome targeting sequences; for review, see Refs. 23 and 26.)

Our studies reveal two previously unknown differences in free protein-GPI metabolism in L. major and T. brucei. First, whereas (free) protein-GPIs accumulate at the ER of T. brucei (Fig. 8), the glycolipids concentrate on the endo-lysosomal system of L. major. Second, organelle-targeting signals on GPIPLCp are deciphered differently by the two trypanosomatids. In T. brucei, unmutated GPI-PLCp is glycosomal (Fig. 8), but in L. major the protein is targeted to the endo-lysosomal system (Fig. 3). Thus, organelle signal peptides from T. brucei may not (always) be recognized in L. major.

A Novel Peptide Motif Targets GPI-PLCp to Endosomes in L. major—Cysteine residues at positions 80, 269, 270, and 273 of GPI-PLCp are part of a novel targeting signal that directs GPI-PLCp to the endo-lysosomal system in L. major (Fig. 3); their mutation to Ser or Ala abolishes GPI-PLCp binding to the organelle. On the contrary, mutation of Cys-184 and Cys-347 did not block targeting of GPI-PLCp to endosomes (Fig. 5).

To determine what might be unique about Cys residues whose mutations redirected GPI-PLCp away from to the endolysosomal system in L. major, an alignment of 20 amino acids surrounding Cys-80 and Cys-273 was performed (Fig. 10). Each of the two Cys residues was found to be part of a motif [CS][CS]-x(0,2)-G-x(1)-C-x(2,3)-S-x(3)-L (PROSITE nomenclature) (64). The motif was not present around those Cys residues (i.e. Cys-184 and Cys-347) whose mutations did not influence endosomal targeting of GPI-PLCp. In the genome of L. major four proteins with the endosome targeting motif were found (Table I). It will be interesting to find out whether those L. major proteins (Table I) bind to endosomes.



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  		FIG. 10.
Alignment (Jotun Hein method) of amino acids surrounding Cys-80 and Cys-273.

 


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  		TABLE I
Leishmania proteins containing a [CS][CS]-x(0,2)-G-x(1)-C-x(2,3)-S-x(3)-L motif

The genome of L. major (www.genedb.org/genedb/leish/index.jsp) was searched for polypeptides containing the motif [CS][CS]-x(0,2)-G-x(1)-C-x(2,3)-S-x(3)-L (PROSITE nomenclature) (64). Proteins with the patterns (bold) are presented together with the surrounding amino acids. Numbers indicate the positions of the peptides in the polypeptide coding sequence.

 

   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant AI53086 (to K. M.-W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Cellular Biology, the University of Georgia, 724 Biological Sciences Bldg., Athens, GA 30602. Tel.: 706-542-3355; Fax: 706-542-4271; E-mail: mensawil{at}cb.uga.edu.

1 The abbreviations used are: GPI, glycosylphosphatidylinositol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ConA, concanavalin A; DAPI, 4,6-diamidino-2-phenylindole dihydrochloride; ER, endoplasmic reticulum; GFP, green fluorescent protein; GlcNH2, glucosamine; GPI-PLCp, glycosylphosphatidylinositol-specific phospholipase C from T. brucei; HGPRT, hypoxanthineguanine phosphoribosyltransferase; Man, mannose; mfVSG, membrane form variant surface glycoprotein from T. brucei; PBS, phosphate-buffered saline; PI, phosphatidylinositol; VSG, variant surface glycoprotein. Back

2 Z. Zheng, R. K. Tweten, and K. Mensa-Wilmot, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We thank Christine Clayton, Jay Bangs, and Buddy Ullman for gifts of antibodies.



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