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ATP酶。 分子伴侣参与蛋白质跨膜转运的过程。 Chirico博士实验室的长期目标是了解分子伴侣如何工作，以及它们如何促进前体蛋白从细胞质到细胞器的翻译后易位。 细胞溶质70 kDa热休克蛋白（Hsp 70）分子伴侣的关注，因为他们在蛋白质折叠和易位中发挥了核心作用。 热休克蛋白70是ATP酶，参与多种涉及蛋白质-蛋白质相互作用的细胞途径。 它们利用ATP的结合和水解能量来调节其寡聚体结构及其与多肽底物的相互作用。 蛋白质调节剂，如DnaJ同源物，调节Hsp 70活性。 在真核细胞质中，Hsp 70和DnaJ同源物足以使变性的蛋白质重折叠，大多数蛋白质从真核细胞输出通过分泌途径。 该途径的第一步是前体蛋白从胞质溶胶转运到内质网。 虽然分泌前蛋白通常是协同转运到内质网中的，但在模式酵母酿酒酵母中，一些分泌前蛋白，例如前α因子原（ppaf），是后分泌转运的。 分泌前蛋白的翻译后易位由胞质、膜结合和内腔蛋白介导。 In S.在酿酒酵母中，胞质组分包括Hsp 70 Ssa 1 p和DnaJ同源物Ydj 1 p。 膜结合组分包括由异源三聚体Sec 61 p复合物和其他四种蛋白质组成的易位装置。 Hsp 70的同源物Kar 2 p是其内腔组分之一，尽管体内外研究表明Ssa 1 p和Ydj 1 p在蛋白质转运中发挥重要作用，但其作用机制尚不清楚。 我们假设Ssa 1 p和Ydj 1 p保持ppaf的易位能力，并将其传递到膜结合易位装置的至少一个组件用于随后的易位。 本研究的目的是阐明Ssa 1 p和Ydj 1 p在ppaf翻译后转运到酵母内质网中的协同作用机制。 这些实验将揭示新的光Hsp 70/DnaJ同源相互作用，因此，也将感兴趣的那些研究伴侣依赖的蛋白质折叠和前体蛋白导入线粒体。 具体目的是确定Ssa 1 p和Ydj 1 p在翻译后易位中的作用，表征涉及Ssa 1 p，Ydj 1 p和ppaf的相互作用，确定伴侣相互作用的序列，并确定伴侣依赖的翻译后易位的拓扑结构。 这项工作将使用生物化学方法完成，包括在体外重建易位到微粒体中。

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.