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

描述(申请人提供)：本项目的长期目标是阐明尾部锚定蛋白(GET)途径的引导进入机制。该途径以尾部锚定(TA)蛋白为靶点，该蛋白被定位于分泌途径，翻译后插入内质网(ER)膜。这一途径的中心成分是伴侣Get3。我们知道，一旦Get3-TA蛋白复合体形成，它们就被招募到内质网膜上插入。除了这个概念框架外，我们仍然对Get3如何找到其TA蛋白底物以及它们通过什么机制插入内质网膜知之甚少。为了更深入地了解GET途径的机制，本项目将确定(1)我们鉴定的上游伴侣复合体如何将带有ER靶向信号的TA蛋白递送到Get3，以及(2)与Get3相关的TA蛋白如何被我们重组的途径的两个膜成分插入到蛋白脂质体中。这些研究将在发芽酵母中进行，因为这种模式生物具有简单的遗传学，在生物化学上易于处理，并且细胞从生物学上可以使用荧光显微镜进行成像。这项拟议的研究将揭示一种保守的靶向途径的机制，该途径使数百种TA蛋白在人类细胞中能够生物发生。从学术角度来看，GET途径很有趣，因为它准确地区分了定位于分泌途径的TA蛋白的膜靶向信号和线粒体TA蛋白的膜靶向信号。此外，该途径使用一种新的膜插入机制，不同于典型的Sec61蛋白转位通道，将跨膜结构域整合到内质网脂双层中。从实用的角度来看，这些研究将阐明尾部锚定的bcl2蛋白如何通过在内质网和线粒体外膜之间的分割来调节细胞凋亡。最后，后生动物Get3同源物Asna 1已成为细胞对顺铂敏感性的关键因素。因此，拟议的研究将提供操纵Asna活性的药理学方法，以最大限度地减少耐药性，并加强现有的顺铂化疗。

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.