Mechanism of Chaperone-Dependent Protein Translocation Into the Endoplasmic Reticulum

分子伴侣是影响蛋白质折叠和构象的蛋白质，通常是三磷酸腺苷酶。伴侣蛋白参与了蛋白质跨膜转运的过程。Chirico博士实验室的长期目标是了解伴侣蛋白是如何起作用的，以及它们是如何促进前体蛋白从细胞质到细胞器的翻译后易位的。细胞内70kda热休克蛋白（Hsp70）分子伴侣在蛋白质折叠和易位中起着核心作用，因此受到关注。hsp70是参与多种涉及蛋白-蛋白相互作用的细胞通路的atp酶。它们利用ATP的结合和水解的能量来调节它们的寡聚结构和它们与多肽底物的相互作用。蛋白质调节剂，如DnaJ同源物，调节Hsp70的活性。在真核细胞质中，Hsp70和兼容的DnaJ同源物足以对变性蛋白进行再折叠。真核细胞输出的大多数蛋白质都经过分泌途径。这一途径的第一步是将前体蛋白从细胞质溶胶转运到内质网。虽然分泌前蛋白通常是共翻译转运到内质网中，但在模型酵母中，一些分泌前蛋白，如前α因子（ppaf），是翻译后转运的。分泌前蛋白的翻译后易位由细胞质蛋白、膜结合蛋白和管腔蛋白介导。在葡萄球菌中，胞质成分包括Hsp70 Ssa1p和DnaJ同源物Ydj1p。膜结合组分包括由异三聚体Sec61p复合体和其他四种蛋白质组成的易位装置。其中一个管腔成分是Hsp70同源物Kar2p。尽管体内和体外研究结果表明，Ssa1p和Ydj1p在蛋白质易位中起着重要的相互作用，但其作用机制仍不清楚。我们假设Ssa1p和Ydj1p维持了ppaf的易位能力，并将其传递给膜结合易位装置的至少一个组成部分进行后续易位。本项目的目的是阐明Ssa1p和Ydj1p在ppaf翻译后易位到酵母内质网中的合作机制。这些实验将为Hsp70/DnaJ同源相互作用提供新的线索，因此，也将引起那些研究伴侣依赖蛋白折叠和前体蛋白导入线粒体的人的兴趣。具体目的是确定Ssa1p和Ydj1p在翻译后易位中的作用，表征Ssa1p、Ydj1p和ppaf的相互作用，确定伴侣相互作用的序列，确定伴侣依赖的翻译后易位的地形。这项工作将使用生化方法完成，包括在体外重组易位到微粒体。

Molecular chaperones are proteins, often ATPases, that influence the folding and conformation of proteins. Chaperones have been implicated in the process whereby proteins are translocated across membranes. The long term goals of Dr. Chirico's laboratory are to understand how chaperones work and how they promote the post-translational translocation of precursor proteins from the cytosol into organelles. Attention is being focused on the cytosolic 70 kDa heat shock protein (Hsp70) molecular chaperones, because they play a central role in protein folding and translocation. Hsp70s are ATPases that participate in a variety of cellular pathways involving protein-protein interactions. They use the energy of binding and hydrolysis of ATP to regulate their oligomeric structure and their interactions with polypeptide substrates. Protein modulators, such as DnaJ homologs, regulate Hsp70 activity. In the eukaryotic cytosol an Hsp70 and a compatible DnaJ homolog are sufficient to refold denatured proteins.Most proteins exported from eukaryotic cells pass through the secretory pathway. The first step in this pathway is the translocation of a precursor protein from the cytosol into the endoplasmic reticulum. Although presecretory proteins are usually translocated into endoplasmic reticulum co-translationally, in the model yeast, Saccharomyces cerevisiae, some presecretory proteins, for example prepro-alpha-factor (ppaf), are translocated post-translationally. Post-translational translocation of presecretory proteins is mediated by cytosolic, membrane-bound, and lumenal proteins. In S. cerevisiae the cytosolic components include the Hsp70 Ssa1p and the DnaJ homolog Ydj1p. The membrane-bound components include a translocation apparatus composed of the heterotrimeric Sec61p complex and four other proteins. One of the lumenal components is the Hsp70 homolog Kar2p.Although results from in vivo and in vitro studies suggest that Ssa1p and Ydj1p play important and interacting roles in protein translocation, the mechanism of action remains largely unknown. We hypothesize that Ssa1p and Ydj1p maintain the translocation competence of ppaf and deliver it to at least one component of the membrane-bound translocation apparatus for subsequent translocation. The goal of this project is to elucidate the mechanism by which Ssa1p and Ydj1p cooperate in the post-translational translocation of ppaf into the endoplasmic reticulum of yeast. These experiments will shed new light on Hsp70/DnaJ homolog interactions and, therefore, will also interest those studying chaperone-dependent protein folding and precursor protein import into mitochondria. The specific aims are to determine the roles of Ssa1p and Ydj1p in post-translational translocation, characterize the interactions involving Ssa1p, Ydj1p, and ppaf, determine the sequence of chaperone interactions, and determine the topography of chaperone-dependent post-translational translocation. The work will be done using biochemical approaches, including in vitro reconstitution of translocation into microsomes.