REGULATION OF GLCNAC-PI DEACETYLATION

这项研究的长期目标是了解 糖基磷脂酰肌醇（GPI）在哺乳动物细胞中的生物合成。这 独特的糖脂被用作膜锚， 不同的蛋白质组的质膜。当GPI生物合成是 被阻断后，这些蛋白质可以合成，但不能正常发挥作用。 功能，因为它们要么在内部降解，要么由细菌分泌。 cell.因此，预期GPI生物合成受到严格调控， 满足糖脂锚的细胞要求。在这一提议中， 我们将研究GPI生物合成的第二步， N-乙酰葡糖胺-磷脂酰肌醇（GlcNAc-PI）脱乙酰化， 形成葡糖胺磷脂酰肌醇（GlcN-PI）。使用无细胞系统， 我们发现这种反应是由GTP刺激的。这种效果是 特异于含鸟苷的核苷酸三磷酸， 在GTP的浓度（最佳为1 mM）下， 相关的初步证据表明，这种调节是通过 通过GTP结合蛋白，需要GTP水解。虽然许多细胞 过程受到GTP的影响，GlcNAc-PI的脱乙酰化代表 这是一种新类型的事件，需要以这种方式进行监管。 我们建议分离GlcNAc-PI缺陷的突变细胞系， 脱乙酰酶，其调节GTP，以及随后的反应， 途径，GlcN-PI酰化形成GlcN-PI（酰基）。这些突变体 将用于表达克隆缺陷基因。推测的蛋白质 从这些克隆获得的序列信息将用于确定 如果这些蛋白质中的任何一个具有共有的GTP结合位点， 针对这些蛋白质的抗体。来自GlcNAc-Pl的微粒体 脱乙酰酶突变体也将与来自突变细胞的微粒体混合 线缺陷的第一步，建立一个新的无细胞系统进行研究 第二步的GTP调节。在初步实验中， 在这种混合膜系统中，我们发现， GlcNAc-PI几乎完全依赖于GTP。GTP的作用 GPI生物合成的第二步将在微粒体中进行研究， 混合膜系统使用GTP耗尽的微粒体，我们将确定 如果GlcNAc-PI脱乙酰化需要该核苷酸或仅仅被刺激 研究了测定反应动力学参数的方法， GTP的存在和不存在将通过以下方式来确定机制： 哪种GTP会影响这个反应最后，介导蛋白质， GTP效应将通过重构该 在已失活的微粒体中刺激。

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.