REGULATION OF GLCNAC-PI DEACETYLATION

这项研究的长期目标是了解 哺乳动物细胞中糖基磷脂酰肌醇(GPI)的生物合成。这 独特的糖脂被用作膜锚，在功能上连接 不同的蛋白质组到质膜上。当GPI生物合成是 被阻断后，这些蛋白质被合成，但不能正常运行 功能，因为它们要么在内部降解，要么由 手机。因此，GPI的生物合成预计将受到严格的监管，以 满足糖脂锚定的细胞要求。在这份提案中， 我们将研究GPI生物合成的第二步，即 N-乙酰氨基葡萄糖-磷脂酰肌醇(GlcNAc-PI)的脱乙酰基 形成氨基葡萄糖-磷脂酰肌醇(GlcN-PI)。使用无牢房系统， 我们发现这个反应是被GTP刺激的。这种效果是 专用于含鸟苷核苷酸的三磷酸和发生 在生理浓度的GTP(最佳为1 mM)下 切合实际。初步证据表明，这一规定是中介的。 通过GTP结合蛋白，需要GTP水解酶。虽然许多手机 过程受GTP的影响，GlcNAc-PI的脱乙酰基代表 以这种方式监管的一种新类型的事件。 我们建议分离GlcNAc-PI中存在缺陷的突变细胞系 脱乙酰酶，其调节的GTP，以及随后的反应在 途径，即GlcN-PI的酰化反应生成GlcN-PI(酰基)。这些变种人 将用于表达克隆缺陷基因。推导出的蛋白质 从这些克隆中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一致的GTP结合位点，并形成 针对这些蛋白质的抗体。来自GlcNAc-Pl的微生物体 脱乙酰酶突变体还将与突变细胞的微生物体混合 在第一步有缺陷的线路上创建一种新的无细胞系统进行研究 GTP监管的第二步。在初步实验中， 在这种混合膜体系中，我们发现了脱乙酰基 GlcNAc-PI几乎完全依赖于GTP。GTP在全球经济一体化中的作用 GPI生物合成的第二步将在微粒体内和 混合膜系统。使用GTP耗尽的微粒子，我们将确定 如果GlcNAc-PI去乙酰化需要这个核苷酸或只是被刺激 靠它。测定反应动力学参数的研究 GTP的存在和不存在将通过以下方式确定其机制 哪个GTP会影响这一反应。最后，调节细胞周期的蛋白质 GTP效应将被识别和分离，通过重组这个 在它已经失活的微粒体内的刺激。

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.