DEFINING COMPONENTS REQUIRED FOR TRANSPORT FROM THE ER

我们已经确定了三个基因之间复杂的遗传交互作用 (BOS1、BET1和SEC22)是蛋白质转运所必需的 酵母中内质网对高尔基复合体的作用目标 我们研究的目的是通过以下方式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。 我们已经开发出一种分析方法，可以重建内质网的运输 通过酵母中的高尔基复合体(Ruohola等人，1988年)。最近，我们 已经用这种方法鉴定了一种功能性的囊泡中间体 从急诊室过渡到高尔基体。与世隔绝 中间层使我们能够对与水泡有关的事件进行排序 队形。可能需要Bos1、Bet1和Sec22蛋白 内质网小泡的萌发或小泡的结合和融合 搬运工到接受者高尔基隔间。中描述的实验 这项提议将利用分子遗传学、细胞分离和 体外试验以解决这一可能性并确定其功能 这些基因产物。 1.对SEC22基因进行测序，获得针对该蛋白的抗体 由这个基因编码的。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BOS1和BET1基因编码可能的膜蛋白。 我们将使用针对这些基因产物准备的抗体来确定它们的 蜂窝内的位置和拓扑。我们还将确定Bos1和 Bet1是一种糖基化的蛋白质，它穿过分泌途径或a 这条路的一部分。3.针对Bos1、Bet1和Sec22的抗体 蛋白质将被用来定义观察到的 基因产品。这些实验将揭示Bos1，Bet1和 Sec22蛋白是复合体的组成部分。或者，这些 蛋白质可能在相同的途径上或平行地在功能上相互作用 小路。此外，我们还将鉴定可能存在的可溶性蛋白质 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BOS1、BET1和SEC22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蛋白阻止囊泡的形成或随后的运输阶段 在试管中。

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.