GPI PHOSPHOLIPASE-C FROM TRYPANOSOMA-BRUCEI CAUSES A GPI-NEGATIVE PHENOTYPE IN LEISHMANIA-MAJOR .1. IMPLICATIONS FOR GPI-NEGATIVE MAMMALIAN-CELLS .2. COMPARTMENTALIZATION OF 2 GPI BIOSYNTHETIC PATHWAYS

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysacchari de) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cy toplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysa ccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associ ated gp63 could not be detected in immunoblots. gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative prot ein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striki ng contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmenta lized within the endoplasmic reticulum, thereby sequestering those int ermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN(1 alpha 6)-myo-inosito l-1-phospholipid, is cytosolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expres sing L. major, secretion of proteins with GPI addition signals, and de pletion of protein-GPI anchor precursors, is similar to that of some p rotein-GPI mutants in higher eukaryotes. These findings have implicati ons for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymph oma.