PROTEIN TRANSLOCATION IN YEAST

拟议研究的主要目标是了解， 从机制上讲，正确的蛋白质是如何被靶向和移位的 在简单真核生物中穿过内质网膜 酿酒酵母。我们将测试我们目前的假设 是否有两条平行的靶向路径，其中一条取决于信号 识别粒子(SRP)，另一种不依赖于SRP。我们的目标是 确定在这两个途径中催化反应的所有基因产物， 并了解它们在体内的分子功能和重要性。 具体地说，i)我们将识别和测序编码所有 酵母SRP和SRP受体的亚基；II)我们将使用体外蛋白 易位检测或部分反应检测要详细分析 它们的作用机制。我们的目标是识别新的功能组件 在靶向和易位并克隆他们的基因方面；iii)我们将使用 随机突变分离SRP、SRP的条件性(cs，ts)突变 受体和新鉴定的成分。这些突变和位置- 预测的GTP结合位点的定向突变，以及在 将使用预测的RNA-RNA、RNA-蛋白质或蛋白质-蛋白质相互作用 在体内和体外分析基因产物的作用；iv)我们将 分离与蛋白质靶向和转位有关的其他基因 使用改进的遗传选择。这些突变将被分析为 他们参与了SRP依赖或非SRP依赖 途径；v)我们将使用对每个途径中突变的抑制分析来 鉴定相互作用的基因产物。 拟议的生化和遗传分析旨在研究一种精确的分子 了解蛋白质的靶向和转位过程。我们希望 了解过程中哪些方面是酵母所特有的，哪些方面是酵母所特有的 可推广到其他真核细胞和细菌。最终，我们 希望通过基因和生化相结合的方法的力量， 我们将确定蛋白质易位的细胞成分 对于运输至关重要的机械，以及那些 调节性的。

The primary objective of the proposed research is to understand, mechanistically, how the correct proteins are targeted to and translocated across the endoplasmic reticulum membrane in the simple eukaryote Saccharomyces cerevisiae. We will test our current hypothesis that there are two parallel targeting pathways, one dependent on the signal recognition particle (SRP), the other one SRP-independent. Our aim is to identify all the gene products catalyzing the reactions in both pathways, and to understand their molecular function and importance in vivo. Specifically, i) we will identify and sequence the genes encoding all subunits of yeast SRP and SRP receptor; ii) we will use in vitro protein translocation assays or assays for partial reactions to analyze in detail their mechanism of action. We aim to identify novel components functioning in targeting and translocation and clone their genes; iii) we will use random mutagenesis to isolate conditional (cs, ts) mutations in SRP, SRP receptor and newly identified components. These mutations and site- directed mutations in predicted GTP binding sites, and in regions of predicted RNA-RNA, RNA-protein or protein-protein interactions will be used to analyze in vivo and in vitro the role of the gene products; iv) we will isolate additional genes involved in protein targeting and translocation using improved genetic selections. Such mutations will be analyzed as to their involvement in either the SRP-dependent or the SRP-independent pathway; v) we will use suppressor analysis of mutations in each pathway to identify interacting gene products. The proposed biochemical and genetic analyses aim at a precise molecular understanding of the protein targeting and translocation process. We wish to learn which aspects of the process are unique to yeast and which aspects can be generalized to other eukaryotic cells and bacteria. Ultimately, we hope that through the power of a combined genetic and biochemical approach, we will identify the cellular constituents of the protein translocation machinery that are essential for translocation, as well as those that are modulatory.