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 de- tected. A peptide motif [CS][CS]-x(0,2)-G-x(1)-C-x(2,3)-Sx(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.

Glycosylphosphatidylinositols (GPIs) are ubiquitous glycolipids in eukaryotes. In the protozoan Leishmania major, GPIs occur "free" or covalently linked to proteins (e.g., gp63) and polysaccharides. While some free GPIs are detected on the plasma membrane, specific sites where GPIs accumulate intracellularly are unknown in most cells, although the glycolipids are synthesized within the secretory system. Herein, we describe a protocol for identifying intracellular sites of GPI accumulation by using alpha-toxin (from Clostridium septicum). Alpha-toxin bound to gp63 and GPIs from L. major. Intracellular binding sites for alpha-toxin were determined in immunofluorescence assays after removal of GPI-anchored macromolecules (e.g., gp63) from the plasma membrane of fixed cells by using detergent. Endosomes were a major site for GPI accretion in L. major. GPI-less gp63 was detected at the endoplasmic reticulum. In studies with live parasites, alpha-toxin killed L. major with a 50% lethal concentration of 0.77 nM.Glycosylphosphatidylinositols (GPIs) exist in three forms in eukaryotes: (i) "free" (i.e., unattached to macromolecules), (ii) tethered to proteins (e.g., gp63 of Leishmania spp.), or (iii) linked to polysaccharides (e.g., lipophosphoglycan of Leishmania) (reviewed in references 21 and 26).Free GPIs in Leishmania major consist of protein-linked GPIs and polysaccharide-linked GPIs. Protein-linked GPIs include biosynthetic intermediates, e.g., glucosamine (GlcNH 2 )-inositol(Ins)-1-phospho-diacylglycerol, mannose (Man)␣1-6Man␣1-4GlcNH 2 -Ins-1-phospho-diacylglycerol, and Man␣1-4GlcNH 2 -Ins-1-phospho-diacylglycerol, and the completed protein anchor ethanolamine-phospho-Man␣1-2Man␣1-6Man␣1-4GlcNH 2 -Ins-1-phospho-diacylglycerol (21, 36). Polysaccharide-linked GPIs (e.g., lipophosphoglycan) contain the Man␣1-3Man␣1-4GlcNH 2 -Ins-1-phospho-diacylglycerol (reviewed inreference 21).Free GPIs are detected on the plasma membrane of vertebrate and trypanosomatid cells (4,22,43,46,54). Other functions aside from the addition to protein and polysaccharide may be envisioned for free intracellular GPIs. In theory, enzymes that digest GPIs (e.g., phospholipases) could produce second messengers (e.g., arachidonic acid or diacylglycerol) that influence cell physiology. Knowing the subregion(s) of cells that GPIs accumulate in and localizing enzymes that catabolize the glycolipids may provide clues about novel functions of intracellular GPIs.We present a general technique for determining the intracellular location of protein-linked GPIs (protein-GPIs) and apply it to L. major. Central to the protocol is the high specificity of alpha-toxin (from Clostridium septicum) for GPIs (25). When added to fixed or permeabilized cells, alpha-toxin recognizes intracellular protein-GPIs. In a GPI-deficient strain of L. major (40), alpha-toxin binding sites diminished dramatically, or were nonexistent, compared to binding sites in control cells. Concanavalin A (ConA), a control lectin, binds GPIs (20, 41, 50, 56, 57), GDP-mannose, and N-glycans...