Transfer ofGlycosyl-Phosphatidylinositol Membrane Anchors to Pblypeptide Acceptors in a Cell-free System

Glycosylinositol phospholipid (GPI) membrane anchors are the sole means of membrane attachment of a large number of cell surface proteins, including the variant surface glycoproteins (VSGs) of the parasitic protozoan, Trypanosoma brucei . Biosynthetic data suggest that GPI-anchored proteins are synthesized with carboxy-terminal extensions that are immediately replaced by GPI, suggesting the existence of preformed GPI species available for transfer to the naYCOSYLINOSITOL phospholipid (GPI)' membrane anchors attach many proteins of varied function to 13eukaryotic cell membranes, almost always to the cell surface (for review see Ferguson and Williams, 1988; Low, 1989 ; Cross, 1990; Thomas et al ., 1990) . It appears that GPI membrane anchors share an evolutionarily conserved linear core structure, consisting ofethanolamine-P-6 man al-2 man al-6 man al-4 G1cN al-6 inositol . This linear sequence links the phospholipid moiety to the protein via an amide bond between ethanolamine and the carboxy-terminal amino acid . Different anchors have both protein and cell-specific side chain modifications and differ in phospholipid structure. Side chain modifications include carbohydrate (galactose, mannose, and GaINAc), extra ethanolamine phosphate, and an extra fatty acid attached to an inositol hydroxyl . Acylation of inositol confers resistance to PI-PLC, an enzyme often used to diagnose GPI anchoring (Roberts et al ., 1988) . The various GPI lipid moieties described include sn-1,2dimyristylglycerol (T. brucei VSG; Ferguson et al ., 1985), 1-alkyl-2-acyl glycerol (human erythrocyte acetylcholinesterase ; Roberts et al ., 1988 and Leishmania majorpromastigote surface protease ; Schneider et al ., 1990), monoacylglycerol (T. brucei procyclic acidic repetitive protein ; Field et al ., 1991), and ceramide (Dictyostelium discoideum contact site A glycoprotein ; Sadeghi et al ., 1988 ; Stadler et al ., 1989) . The functional significance of these structural variations is unclear. Mature GPI-anchored proteins lack a carboxy-terminal peptide that is encoded in the cDNA sequence . Putative precursor proteins that contain this extension have been ob1 . Abbreviations used in this paper: GPI, glycosyl-phosphatidyinositol ; Inos, inositol ; pCMPSA, sodium p-chloromercuriphenylsulphonate; PLC, phospholipase C ; VSG, variant surface glycoprotein . © The Rockefeller University Press, 0021-9525/91/07/61/11 $2 . 00 TheJournal of Cell Biology, Volume 114, Number 1, July 199161-71 scent protein in the ER. Candidate precursor glycolipids having a linear sequence indistinguishable from the conserved core structure found on all GPI anchors, have been characterized in T. brucei . In this paper we describe the transfer of three GPI variants to endogenous VSG in vitro . GPI addition is not reduced by inhibitors of protein synthesis and does not require ATP or GTP, consistent with a transpeptidation mechanism . served for neural cell adhesion molecule, N-CAM 120 (He et al ., 1987), and alkaline phosphatase (Takami et a] ., 1988) . Biosynthetic studies suggest that the carboxy-terminal sequence is replaced within 1-5 min of completion of protein synthesis (Bangs et al ., 1985 ; Ferguson et al ., 1986 ; Conzelmann et al ., 1987; He et al ., 1987) . The rapidity of this modification suggested the existence of a preformed precursor glycolipid that could be transferred en bloc to the newly synthesized protein concomitant with or after cleavage ofthe carboxy-terminal peptide extension . Recent studies using a yeast secretory mutant (sec 18) support an endoplasmic reticulum location for the GPI addition reaction (Conzelmann et al ., 1988) . The abundance of VSG in T. brucei makes this protozoan parasite a good model for the study ofGPI-anchor structure and biosynthesis . Two glycolipids, P2 and P3 (Menon et al ., 198ßa [presumably identical to lipids A and C; Krakow et al ., 1986]), with characteristics expected of GPI anchor precursors have been identified in this parasite. P2 is PI-PLC sensitive and consists of a linear sequence identical to the conserved core sequence found on all GPI anchors (Menon et al ., 19ß8a ; Mayor et al ., 1990b) . P3 is PI-PLC resistant due to the acylation of the inositol hydroxyl by a fatty acid but is otherwise identical to P2 (Krakow et a] ., 1989 ; Mayor et al ., 1990a) . The biosynthesis of these glycolipids has been studied in vivo (Schwarz et al ., 1988) and in vitro (Menon et al ., 19ß8b ; Masterson et al ., 1990 ; Doering et al ., 1989 ; Masterson et al ., 1989 ; Menon et al ., 1990b) . The simplest model (summarized in Fig . 1) suggested by the available data is that the lipids are assembled by the sequential glycosylation of PI followed by the addition ofphosphoethanolamine . A peculiar feature of trypanosome GPI biosynthesis is the observation of heterogenously acylated forms of P2 and P3, 61 on M ay 0, 2017 D ow nladed fom Published July 1, 1991