A Dissection of the Yeast ER Translocation Machine

内质网（ER）接收、折叠和分类从真核细胞分泌的蛋白质或靶向其他细胞内细胞器的蛋白质。 为了促进这些蛋白质输入或易位到ER中，与ER相关的无数细胞质和内腔因子被容纳在多蛋白质机器中。 这些机器中包括分子伴侣，分子伴侣是在细胞中蛋白质折叠和降解过程中发挥重要作用的因子，并且已被证明可以在体外调节多蛋白复合物。损害分子伴侣的活性会阻止某些蛋白质进入ER。 这些因素促进蛋白质易位的机制知之甚少，部分原因是当体内单个分子伴侣的活性受损时，许多细胞过程中会发生多效性缺陷。 为了避免这个问题，以了解伴侣蛋白对ER输入的具体影响，已开发了体外重构系统。 随后从模式酵母酿酒酵母中分离新的伴侣突变体，并在体外分析中分析它们，从而可以从分子上详细说明支持蛋白质易位所需的伴侣活性。酵母中蛋白质易位所需的伴侣包括分别位于内质网腔和细胞质中的hsp 70 ATP酶BiP和Ssa 1p，DnaJ同源物Sec63p和Ydj1p分别位于管腔和细胞质中。 Hsp70 s和DnaJ蛋白在所有物种中都具有协同作用，最近对新的显性致死BiP突变体的分析表明，ATP依赖的构象变化是支持BiP和Sec63 p相互作用的必要条件，也是驱动蛋白质进入内质网的必要条件。 该项目包括生物化学研究，以确定为什么显性出现在这些突变体。 这些实验的结果将表明BiP在蛋白质输入过程中发挥的重要作用，并可能验证或反驳现有的假设，描述蛋白质易位过程中分子伴侣的作用。 胞质伴侣Ssap1和Ydjp1促进易位的机制以及Ssa1p在导入过程中对ATP水解的需求仍然是神秘的。 为了解开这个谜团，在之前的资助期间，构建了新型ATP酶缺陷的ssa1突变体，并纯化了所得的蛋白质。 在这个更新项目中，突变体和野生型蛋白质将进行一系列已建立的生化测试，以阐明Ssa1p ATP酶活性如何与蛋白质易位偶联。 Ydj1p在核糖体上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，其中之一是编码未表征的hsp110伴侣Sse1p的基因。 将确定该分子伴侣对体内和体外易位和翻译反应的贡献。 由于它们在细胞过程中的重要而多样的作用，分子伴侣功能的研究已经产生了关于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的线索。 使用新的生物化学，细胞生物学和遗传学工具的伴侣蛋白功能的持续分析无疑将继续有助于了解这些和其他细胞功能。

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.