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细胞内细胞器的蛋白质。为了促进这些蛋白质的输入或转运到内质网，与内质网相关的无数细胞质和腔因子被安置在多蛋白质机器中。这些机器中包括分子伴侣，这是一种在细胞内蛋白质折叠和降解过程中发挥重要作用的因子，已被证明在体外调节多蛋白质复合体。破坏分子伴侣的活性可以阻止一些蛋白质进入内质网。这些因素促进蛋白质转位的机制尚不清楚，部分原因是当单个伴侣的活性在体内受到影响时，许多细胞过程中会发生多营养缺陷。为了解决这一问题，为了了解伴侣蛋白对内质网导入的具体作用，人们开发了重组的体外系统。随后从模型酵母中分离出新的伴侣突变体，并对其进行体外分析，从而获得了支持蛋白质转运所需的伴侣活性的分子细节。酵母中蛋白质转运所需的伴侣分别包括位于ER腔和细胞质中的HSP70 ATPase Bip和Ssa1p，以及分别位于腔和细胞质中的DNAJ同系物Sec63p和Ydj1p。Hsp70和Dna J蛋白在所有已被鉴定的物种中协同并促进许多细胞过程。最近对新的显性致死Bip突变体的分析表明，依赖于ATP的构象变化对于支持Bip和Sec63p之间的相互作用是必要的，并且对于推动蛋白质进入内质网是必要的。该项目包括生化研究，以确定为什么在这些突变体中出现显性。这些实验的结果将表明(S)BiP在蛋白质输入过程中扮演的重要角色，并可能验证或驳斥现有的描述蛋白质转运过程中分子伴侣作用的假说。胞质伴侣Ssap1和Ydjp1促进易位的机制以及Ssa1p在进口过程中对ATP水解的需求仍然是个谜。为了解开这个谜团，构建了新的ATPase缺陷ssa1突变体，并在之前的资助期间纯化了由此产生的蛋白质。在这个更新项目中，突变型和野生型蛋白将接受一系列既定的生化测试，以阐明Ssa1p ATPase活性如何与蛋白质易位相关联。Ydj1p在核糖体上的转位和蛋白质翻译过程中扮演着不明确的角色，因此获得了ydj1p温度敏感突变体的多拷贝抑制物，其中之一是一个编码未知hsp110伴侣的基因Sse1p。这个伴侣对体内和体外的移位和翻译反应的贡献将被确定。由于它们在细胞过程中扮演着重要的、不同的角色，对分子伴侣功能的研究已经提供了关于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。使用新的生化、细胞生物学和遗传工具继续分析伴侣功能无疑将继续有助于理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.