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定位和转移的， 在简单的真核生物中， 酿酒酵母 我们将测试我们目前的假设， 是两个平行的靶向通路，一个依赖于信号， 识别粒子（SRP），另一个SRP独立。 我们的目标是 鉴定催化两种途径中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。 具体来说，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质 易位测定或部分反应测定以详细分析 他们的行动机制。 我们的目标是识别新的组件功能 在靶向和易位和克隆它们基因方面; iii）我们将使用 随机诱变以分离SRP中的条件（cs，ts）突变，SRP 受体和新发现的成分。 这些突变和位点- 预测的GTP结合位点中的定向突变，以及 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 分离参与蛋白质靶向和易位的其它基因 使用改良的基因选择。 这些突变将被分析， 他们参与了SRP依赖或SRP独立 途径; v）我们将使用每个途径中突变的抑制基因分析， 识别相互作用的基因产物。 所提出的生化和遗传分析的目的是在一个精确的分子 了解蛋白质靶向和转运过程。 我们希望 了解酵母在这一过程中的哪些方面是独特的， 可以推广到其他真核细胞和细菌。 最终我们 希望通过基因和生物化学相结合的方法， 我们将鉴定蛋白质易位的细胞成分 对于易位至关重要的机器，以及那些 调节的

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.