INTERACTION OF YPT1 WITH ITS ACCESSORY PROTEINS

我们将纯化香叶基香叶基转移酶(GGTase-II) 异戊二烯基化物Ypt1和Sec4以及定义这些GTP的结构域- 与这种酶相互作用的结合蛋白。添加了一个配基 到转移酶的一个亚单位上，以便于纯化这一 酶复合体的亲和层析。纯化的亚基将 使用各种分子方法进行克隆，克隆的基因 将在体外进行诱变。的表型后果 致突变GGTase-II的每个亚基也将被分析。 此外，我们正在鉴定一个突变体的抑制子(BTS1)，该突变体 在转移酶的一个亚基上有缺陷。BTS1可以编码 GGTase-II或另一种戊基转移酶的一种成分，作用于 这条路。了解GGTase-II的特异性， 关于法尼基转移酶和GGTase-I，可能具有治疗作用 在治疗癌症和心脏病方面的价值。 Ypt1被GGTase-II修饰后，它结合到膜上，在那里它 与其他与其共同发挥作用的蛋白质相互作用 调节内质网向高尔基体转运的晚期。目前，我们正在 鉴定两种膜蛋白，它们的过度表达可以补偿 对于Ypt1功能的丧失。将进行交联性研究 以确定这些蛋白质是否与这种异构体相互作用 GTP结合蛋白。此外，我们还设计了一种基因筛查 识别编码与之相互作用的辅助蛋白的新基因 Ypt1.最终，这些研究将阐明这一小小的 GTP结合蛋白在调节内质网之间的囊泡运输中的作用 和高尔基情结。

We will purify the geranylgeranyltransferase (GGTase-II) that isoprenylates Ypt1 and Sec4 as well as define the domains of these GTP- binding proteins that interact with this enzyme. A ligand has been added onto a subunit of the transferase to facilitate the purification of this enzyme complex by affinity chromatography. The purified subunits will be cloned using a variety of molecular approaches, and the cloned genes will be mutagenized in vitro. The phenotypic consequences of mutagenizing each subunit of the GGTase-II will also be analyzed. Additionally, we are characterizing a suppressor (BTS1) of a mutant that is defective in one of the subunits of the transferase. BTS1 may encode a component of the GGTase-II or another prenyltransferase that acts on this pathway. Understanding the specificity of the GGTase-II, with respect to the farnesyltransferase and GGTase-I, may have therapeutic value in the treatment of cancer and heart disease. After Ypt1 is modified by the GGTase-II, it binds to membranes where it interacts with other proteins that function in conjunction with it to mediate a late stage in ER to Golgi transport. Currently, we are characterizing two membrane proteins, whose overexpression compensates for the loss of Ypt1 function. Crosslinking studies will be performed to determine if these proteins interact with the prenylated form of this GTP-binding protein. In addition, we have devised a genetic screen to identify new genes that encode the accessory proteins that interact with Ypt1. Ultimately, these studies will elucidate the role that this small GTP-binding protein plays in mediating vesicular traffic between the ER and Golgi complex.