A Dissection of the Yeast ER Translocation Machine

内质网（ER）接收、折叠和分类真核细胞分泌的蛋白质或靶向其他胞内细胞器的蛋白质。为了促进这些蛋白质进入内质网的进口或易位，与内质网相关的无数细胞质和管腔因子被安置在多蛋白机器中。这些机器包括分子伴侣，在细胞内蛋白质折叠和降解过程中起重要作用的因素，并已被证明在体外调节多蛋白质复合物。破坏分子伴侣的活性会阻止一些蛋白质进入内质网。这些因子促进蛋白质易位的机制尚不清楚，部分原因是当体内单个伴侣的活性受损时，许多细胞过程中的多营养缺陷就会发生。为了避免这一问题，以了解伴侣蛋白对内质网进口的具体影响，已经开发了体外重建系统。随后从模式酵母（酿酒酵母）中分离出新的伴侣突变体，并对其进行体外分析，从而获得支持蛋白质易位所需的伴侣活性的分子细节。酵母蛋白易位所需的伴侣包括内质网管腔和细胞质中的hsp70 atp酶BiP和Ssa1p，以及细胞质和管腔中的DnaJ同源物Sec63p和Ydj1p。已知hsp70和DnaJ蛋白在所有已确定的物种中合作并促进许多细胞过程。最近对新的显性致死性BiP突变体的分析表明，atp依赖的构象变化对于支持BiP和Sec63p之间的相互作用是必要的，并且是驱动蛋白质进入内质网络的必要条件。该项目包括生化研究，以确定为什么这些突变体中出现显性。这些实验的结果将表明BiP在蛋白质导入过程中所起的重要作用，并可能验证或反驳描述分子伴侣在蛋白质易位过程中作用的现有假设。胞质伴侣Ssap1和Ydjp1促进转运的机制以及Ssa1p在进口过程中对ATP水解的要求仍然是一个谜。为了解开这个谜团，新的atpase缺陷ssa1突变体被构建，并在之前的资助期内纯化了产生的蛋白质。在这个更新项目中，突变型和野生型蛋白将进行一系列已建立的生化试验，以阐明Ssa1p atp酶活性如何与蛋白质易位耦合。Ydj1p在核糖体的易位和蛋白质翻译过程中起着不明确的作用；因此，获得了ydj1p温度敏感突变体的多拷贝抑制子，其中一个是编码未表征的hsp110伴侣子Sse1p的基因。这种伴侣对体内和体外易位和翻译反应的贡献将被确定。由于分子伴侣在细胞过程中发挥着重要而多样的作用，对分子伴侣功能的研究已经为DNA复制、蛋白质降解和磷酸化以及细胞周期控制的基本机制提供了线索。使用新的生化、细胞生物学和遗传学工具对伴侣蛋白功能的持续分析无疑将继续有助于对这些和其他细胞功能的理解。

The endoplasmic reticulum (ER) receives, folds, and sorts proteins that are secreted from the eukaryotic cell or that are targeted to other intracellular organelles. To facilitate the import, or translocation, of these proteins into the ER, a myriad of cytoplasmic and lumenal factors associated with the ER are housed in multi-protein machines. Included in these machines are molecular chaperones, factors that play vital roles during protein folding and degradation in the cell and that have been shown to regulate multi-protein complexes in vitro. Compromising the activities of molecular chaperones arrests the import of some proteins into the ER. The mechanism by which these factors facilitate protein translocation is poorly understood, in part because pleiotrophic defects in many cellular processes occur when the activity of a single chaperone is compromised in vivo. To circumvent this problem in order to understand the specific effects of chaperones on ER import, reconstituted in vitro systems have been developed. The subsequent isolation of novel chaperone mutants from the model yeast, Saccharomyces cerevisiae, and their analysis in such in vitro assays, permits a molecular detailing of which chaperone activities are required to support protein translocation.Chaperones required for protein translocation in yeast include the hsp70 ATPases BiP and Ssa1p in the ER lumen and cytoplasm, respectively, and the DnaJ homologues Sec63p and Ydj1p in the lumen and cytoplasm, respectively. Hsp70s and DnaJ proteins are known to cooperate and facilitate a number of cellular processes in all species in which they have been identified.A recent analysis of new dominant lethal BiP mutants indicates that an ATP-dependent conformational change is imperative to support the interaction between BiP and Sec63p and is necessary to drive protein import into the ER. This project includes biochemical studies to determine why dominance arises in these mutants. The results of these experiments will indicate which essential role(s) BiP plays during protein import, and may either validate or refute existing hypotheses describing the action of molecular chaperones during protein translocation. The mechanism by which the cytosolic chaperones Ssap1 and Ydjp1 facilitate translocation and the requirement for ATP hydrolysis by Ssa1p during import remains mysterious. To unravel this mystery, novel ATPase-defective ssa1 mutants were constructed and the resulting proteins purified during the preceding funding period. In this renewal project, the mutant and wild type proteins will be subjected to a battery of established biochemical tests to elucidate how Ssa1p ATPase activity is coupled to protein translocation. Ydj1p plays ill-defined roles during both translocation and protein translation on ribosomes; therefore multicopy suppressors of a ydj1p temperature sensitive mutants were obtained, one of which was a gene encoding an uncharacterized hsp110 chaperon, Sse1p. The contribution of this chaperone to the translocation and translation reactions, both in vivo and in vitro, will be determined. Because of their vital, diverse roles in cellular processes, studies of molecular chaperone function have yielded clues regarding the fundamental mechanisms underlying DNA replication, protein degradation and phosphorylation, and cell cycle control. The continued analysis of chaperone functions using novel biochemical, cell biological and genetic tools will undoubtedly continue to contribute to the understanding of these and other cellular functions.