DEFINING COMPONENTS REQUIRED FOR TRANSPORT FROM THE ER

我们已经确定了三个基因之间的复杂的遗传相互作用 (BOS1，BET 1和SEC 22）所需的蛋白质转运从 在酵母菌中内质网与高尔基复合体的连接 我们研究的一个目的是通过以下方式来定义这些相互作用的性质： 对这些基因及其产物进行详细分析。 我们已经开发了一种检测方法， 通过酵母中的高尔基复合体（Ruohola等人，1988年）。 最近我们 已经使用该测定法鉴定了一种功能性囊泡中间体， 从内质网转移到高尔基体。 这种隔离 中间体使我们能够对囊泡的事件进行排序， 阵 Bos 1、Bet1和Sec22蛋白可能是细胞增殖所必需的。 囊泡从ER出芽或囊泡的结合和融合 载体到受体高尔基室。 中描述的实验 这项建议将利用分子遗传学、细胞分级分离和 体外试验，以解决这种可能性，并确定功能 这些基因产物。 1. 我们将对SEC22基因进行测序，并获得针对该蛋白的抗体 由这个基因编码。 抗Sec22抗体将用于评估 Sec22蛋白的细胞内定位。2. DNA序列分析 预测BOS 1和BET 1基因编码推定的膜蛋白。 我们将使用针对这些基因产物制备的抗体来确定它们的 细胞内位置和拓扑结构。 我们还将确定Bos1和 Bet1是糖基化的蛋白质，其穿过分泌途径或 这条路的一部分。3. Bos1、Bet1和Sec22抗体 蛋白质将被用来定义这些蛋白质之间观察到的相互作用。 基因产品 这些实验将揭示Bos1、Bet1和 Sec22蛋白是复合物的组分。 或者，这些 蛋白质可在相同途径或平行途径上功能性地相互作用 途径。 此外，我们将鉴定可溶性蛋白质， 与Bos1和Bet1蛋白的亲水结构域相互作用。4.我们 将在哺乳动物中筛选BOS 1、BET 1和SEC 22基因的同源物， 细胞5.我们将确定是否制备了针对Bos1、Bet1和 Sec22蛋白阻断囊泡形成或随后的运输阶段 体外

We have identified an elaborate genetic interaction among three genes (BOS1, BET1 and SEC22) required for the transit of proteins from the lumen of the ER to the Golgi complex in the yeast Saccharomyces The goal of our research is to define the nature of these interactions by performing a detailed analysis of these genes and their products. We have developed an assay that reconstitutes transport from the ER through the Golgi complex in yeast (Ruohola et al., 1988). Recently, we have used this assay to identify a functional vesicular intermediate in transit from the ER to the Golgi apparatus. The isolation of this intermediate has enabled us to order events with respect to vesicle formation. The Bos1, Bet1 and Sec22 proteins may be required for the budding of vesicles from the ER or the binding and fusion of vesicular carriers to the acceptor Golgi compartment. The experiments described in this proposal will utilize molecular genetics, cell fractionation and the in vitro assay to address this possibility and to determine the function of these gene products. 1. We will sequence the SEC22 gene and obtain antibody to the protein encoded by this gene. The anti-Sec22 antibody will be used to assess the intracellular location of the Sec22 protein. 2. DNA sequence analysis predicts that the BOS1 and BET1 genes encode putative membrane proteins. We will use antibodies prepared to these gene products to ascertain their intracellular location and topology. We will also determine if Bos1 and Bet1 are glycosylated proteins that traverse the secretory pathway or a portion of this pathway. 3. Antibodies to the Bos1, Bet1, and Sec22 proteins will be used to define the interactions observed among these genes products. These experiments will reveal whether the Bos1, Bet1 and Sec22 proteins are components of a complex. Alternatively, these proteins may functionally interact on the same pathway or parallel pathways. In addition, we will identify soluble proteins that may interact with the hydrophilic domain of the Bos1 and Bet1 proteins. 4. We will screen for homologues of the BOS1, BET1 and SEC22 genes in mammalian cells. 5. We will determine if antibodies prepared to the Bos1, Bet1 and Sec22 proteins block vesicle formation or subsequent stages of transport in vitro.