Mechanistic analysis of post-translation membrane protein insertion into the ER.

翻译后膜蛋白插入内质网的机制分析。

• 批准号: 8450736
• 负责人: Vladimir Denic
• 金额: $ 30.33万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8450736
• 关键词: ATP phosphohydrolase; Address; Affinity; Animal Model; Apoptosis; Binding; Biogenesis; Cell physiology; Cells; Cisplatin; Complement; Complex; Data; Drug resistance; Drug usage; Endoplasmic Reticulum; Fluorescence Microscopy; Genetic; Goals; Homologous Gene; Human; Image; In Vitro; Light; Lipid Bilayers; Location; Malignant Neoplasms; Membrane; Membrane Proteins; Mitochondria; Molecular Chaperones; Molecular Conformation; Molecular Machines; Monitor; Mutation; Nucleotides; Outer Mitochondrial Membrane; Pathway interactions; Pharmaceutical Preparations; Play; Point Mutation; Protein Binding; Protein translocation; Proteins; Recruitment Activity; Regulation; Resistance; Role; Saccharomycetales; Signal Transduction; Sorting - Cell Movement; Specific qualifier value; Structure; System; Tail; Testing; Translations; Transmembrane Domain; Work; chemotherapy; dimer; improved; in vivo; insight; interest; mutant; novel; protein complex; proteoliposomes; reconstitution; research study; tool

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the mechanism of the Guided Entry of Tail-anchored proteins (GET) pathway. This pathway targets tail-anchored (TA) proteins destined for the secretory pathway for post-translational insertion into the endoplasmic reticulum (ER) membrane. The central component of this pathway is the chaperone Get3. We know that once Get3-TA protein complexes are formed they are recruited to the ER membrane for insertion. Beyond this conceptual framework, we still know very little about how Get3 finds its TA protein substrates and by what mechanism they are inserted into the ER membrane. To gain a deeper mechanistic understanding of the GET pathway, this project will determine (1) how an upstream chaperone complex that we identified delivers TA proteins with ER-targeting signals to Get3, and (2) how TA proteins associated with Get3 are inserted by two membrane components of the pathway that we have reconstituted into proteoliposomes. These studies will be performed in budding yeast because this model organism has facile genetics, is biochemically tractable, and cell biologically accessible to imaging using fluorescence microscopy. The proposed studies will reveal the mechanism of a conserved targeting pathway that enables the biogenesis of hundreds of TA proteins in human cells. From an academic standpoint, the GET pathway is interesting because it accurately distinguishes the membrane targeting signals of TA proteins destined for the secretory pathway form those of mitochondrial TA proteins. Furthermore, this pathway uses a novel membrane insertion machinery, distinct from the canonical Sec61 protein translocation channel, to integrate transmembrane domains into the ER lipid bilayer. From a practical standpoint, these studies will shed light on how tail-anchored Bcl2 proteins regulate apoptosis by partitioning between the ER and the mitochondrial outer membrane. Lastly, Asna1, the metazoan Get3 homolog, has emerged as a critical factor for cellular sensitivity to cisplatin. Thus, the proposed studies will inform pharmacological approaches for manipulating Asna1 activity to minimize drug resistance and enhance existing cisplatin chemotherapies.

描述（由申请人提供）：该项目的长期目标是阐明尾锚蛋白（GET）途径的指导进入的机制。该途径靶向尾巴锚定的（TA）蛋白，该蛋白注定要用于分泌后途径，用于将翻译后插入到内质网（ER）膜中。该途径的中心部分是伴侣GET3。我们知道，一旦形成了GET3-TA蛋白复合物，它们就会被募集到ER膜进行插入。除了这个概念框架之外，我们仍然对GET3如何找到其TA蛋白底物以及通过将它们插入什么机制插入ER膜中的方式知之甚少。为了更深入地了解GET途径，该项目将确定（1）我们确定的上游伴侣复合物如何将具有ER靶向信号的TA蛋白传递给GET3，以及（2）与Get3相关的TA蛋白如何由我们对蛋白质蛋白蛋白蛋白蛋白的两个途径组件插入的TA蛋白如何插入。这些研究将在萌芽的酵母中进行，因为该模型有机体具有容易的遗传学，在生物化学上是可触犯的，并且在生物学上可以使用荧光显微镜进行成像。拟议的研究将揭示一种保守的靶向途径的机制，该途径能够在人类细胞中数百种TA蛋白的生物发生。从学术的角度来看，GET道路很有趣，因为它准确区分了针对分泌途径的TA蛋白的膜靶向信号，形成了线粒体TA蛋白的膜。此外，该途径使用一种新型的膜插入机械，与规范的SEC61蛋白易位通道不同，将跨膜结构域整合到ER脂质双层中。从实际的角度来看，这些研究将阐明尾锚的Bcl2蛋白如何通过在ER和线粒体外膜之间分配来调节凋亡。最后，Metazoan GET3同源物ASNA1已成为对顺铂敏感性的关键因素。因此，拟议的研究将为操纵ASNA1活性的药理方法提供信息，以最大程度地减少耐药性并增强现有的顺铂化学疗法。