Stimulation of glycosylphosphatidylinositol biosynthesis in mammalian cell-free systems by GTP hydrolysis: evidence for the involvement of membrane fusion

The second step in glycosylphosphatidylinositol (GPI) biosynthesis, the dea cetylation of GlcNAc-phosphatidylinositol (GlcNAc-PI), has been shown to be stimulated by GTP hydrolysis [Stevens (1993) J. Biol. Chem. 268, 9718-9724 ]. We have now developed a system to study this regulation that uses micros omes from cells defective in the first step in GPI biosynthesis (class A, C and H lymphoma mutants) and the second reaction in the pathway (G9PLAP.85) . With this mixed-microsome system, the deacetylation of GlcNAc-PI was almo st completely dependent on GTP hydrolysis. Because GlcNAc-PI synthesized by the G9PLAP.85 microsomes cannot readily move to the first-step-mutant micr osomes to be deacetylated, this result indicated that the role of GTP was t o facilitate the 'apparent' transfer of this substrate between membrane ves icles. The microsomes could be stably preactivated by pretreatment with GTP before GPI biosynthesis was initiated, indicating that fusion was the most likely mechanism for this regulation. GlcNAc-PI deacetylation could also b e stably preactivated in EL4 microsomes, suggesting that fusion also occurr ed in wild-type membranes. Some differential localization of the GlcNAc-PI synthetic and deacetylation activities with the endoplasmic reticulum was f ound. Therefore fusion seems to stimulate GPI biosynthesis in mammalian mic rosomes by bringing together the first two enzymes in the pathway in the sa me membrane vesicle.