The long-term goal of this research is to understand the regulation of glycosylphosphatidyl-inositol (GPI) biosynthesis in mammalian cells. This unique glycolipid is used as a membrane anchor to attach a functionally diverse group of proteins to the plasma membrane. When GPI biosynthesis is blocked, these proteins are synthesized but cannot perform their normal function because they are either degraded internally or secreted by the cell. Therefore, GPI biosynthesis is expected to be tightly regulated to meet the cellular requirements for glycolipid anchors. In this proposal, we will study the regulation of the second step in GPI biosynthesis, the deacetylation of N-acetylglucosamine-phosphatidylinositol (GlcNAc-PI) to form glucosamine-phosphatidylinositol (GlcN-PI). Using a cell-free system, we have found that this reaction is stimulated by GTP. This effect is specific for the guanosine-containing nucleotide triphosphate and occurs at a concentration of GTP (optimal at 1 mM) that is physiologically relevant. Preliminary evidence suggests that this regulation is mediated by a GTP-binding protein and requires GTP hydrolysis. While many cellular processes are affected by GTP, the deacetylation of GlcNAc-PI represents a new type of event to be regulated in this manner. We propose to isolate mutant cell lines with defects in the GlcNAc-PI deacetylase, its regulation by GTP, and the subsequent reaction in the pathway, the acylation of GlcN-PI to form GlcN-PI (acyl). These mutants will be used to expression clone the defective genes. The deduced protein sequence information obtained from these clones will be used to determine if any of these proteins have a consensus GTP binding site and to develop antibodies against these proteins. Microsomes from the GlcNAc-Pl deacetylase mutant will also be mixed with microsomes from a mutant cell line defective in the first step to create a new cell-free system to study the GTP regulation of the second step. In preliminary experiments with this mixed membrane system, we have found that the deacetylation of GlcNAc-PI is almost completely dependent on GTP. The role of GTP in the second step of GPI biosynthesis will investigated in the microsomes and mixed membrane systems. Using GTP-depleted microsomes, we will determine if GlcNAc-PI deacetylation requires this nucleotide or is just stimulated by it. Studies to measure the kinetic parameters of the reaction m the presence and absence of GTP will be done to determine the mechanism by which GTP affects this reaction. Finally, the protein that mediates the GTP effect will be identified and isolated by reconstituting this stimulation in microsomes in which it had been inactivated.
| Stevens, V L; Zhang, H; Kristyanne, E S (1999) Stimulation of glycosylphosphatidylinositol biosynthesis in mammalian cell-free systems by GTP hydrolysis: evidence for the involvement of membrane fusion. Biochem J 341 ( Pt 3):577-84 |
| Gordon, V M; Nelson, K L; Buckley, J T et al. (1999) Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors. J Biol Chem 274:27274-80 |
| Nakamura, N; Inoue, N; Watanabe, R et al. (1997) Expression cloning of PIG-L, a candidate N-acetylglucosaminyl-phosphatidylinositol deacetylase. J Biol Chem 272:15834-40 |
