ACYLATION OF GLUCOSAMINYL PHOSPHATIDYLINOSITOL REVISITED - PALMITOYL-COA DEPENDENT PALMITOYLATION OF THE INOSITOL RESIDUE OF A SYNTHETIC DIOCTANOYL GLUCOSAMINYL PHOSPHATIDYLINOSITOL BY HAMSTER MEMBRANES PERMITS EFFICIENT MANNOSYLATION OF THE GLUCOSAMINE RESIDUE

Two critical steps in the assembly of yeast and mammalian glycosylphos phatidylinositol (GPI) anchor precursors are palmitoylation of the ino sitol residue and mannosylation of the glucosamine residue of the gluc osaminyl phosphatidylinositol (GlcN alpha-PI) intermediate. Palmitoyla tion has been reported to be acyl-CoA dependent in yeast membranes (Co stello, L. C., and Orlean, P. (1992) J. Biol. Chem. 267, 8599-8603) bu t strictly acyl-CoA independent in rodent membranes (Stevens, V. L., a nd Zhang, H. (1994) J. Biol. Chem. 269, 31397-31403), and thus poorly conserved. In addition, it was suggested that acylation must precede m annosylation in both yeast (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 276, 8599-8603) and rodent (Urakaze, M., Kamitani, T., DeG asperi, R., Sugiyama, E., Chang, H.-M., Warren, C. D., and Yeh, E. T. H. (1992) J. Biol. Chem. 267, 6459-6462) cells because GlcN alpha-acyl -PI accumulates in vivo when mannosylation is blocked. However, GlcN a lpha-acyl-PI accumulation would also be expected if mannosylation and acylation were independent of each other. These issues were addressed by the use of a synthetic dioctanoyl GlcN alpha-PI analogue (GlcN alph a-PI(C8)) as an in vitro substrate for GPI-synthesizing enzymes in Chi nese hamster ovary cell membranes. GlcN alpha-PI(C8) was acylated in a n manner requiring acyl-CoA. Thus, the process involving acyl-CoA repo rted for yeast has been conserved in mammals. Furthermore, both GlcN a lpha-PI(C8) and GlcN alpha-acyl-PI(C8) could be mannosylated in vitro, but mannosylation of the latter was significantly more efficient. Thi s provides direct support for the earlier suggestion that acylation pr ecedes mannosylation in rodents cells. A similar result was also obser ved with the Saccharomyces cerevisiae mannosyltransferase. In contrast , it has been reported that mannosylation of endogenous GlcN alpha-PI by Trypansoma brucei membranes occurs without prior acylation. The sam e result was obtained with GlcN alpha-PI(C8), confirming that the mann osyltransferase of trypanosomes is divergent from those in yeasts and rodents.