Regulation of endoytosis by clathrin light chain.

描述(由申请人提供)：我们实验室的长期目标之一是了解网状蛋白轻链是如何调节内吞过程的。这项建议是在酿酒酵母模型系统中研究LC，该模型系统提供了敲除、替换或以其他方式改变感兴趣的基因的能力，以及用于相互作用分子或交叉路径的筛子的可用性。很早就知道，酵母中网状蛋白的重链(HC)或轻链(LC)的缺失会导致α-因子摄取的缺陷和胞内斑块的进展。我们的实验室是第一个认识到LC可以抑制HC零的内吞缺陷的实验室之一。此外，我们还证明了这种抑制是由LC(LC-NT)的N-末端(氨基酸1-143)所介导的。以LC为诱饵的酵母双杂交鉴定了与PIP2结合的Talin样蛋白Sla2(哺乳动物内吞适配器Hip1/Hip1 R的酵母同源物)是一个新的结合伙伴，并进一步将这种相互作用定位为LC-NT。我们认为LC抑制HC缺失涉及LC对Sla2的调节。因此，这项建议的主要目的是确定LC对Sla2的调节如何促进内吞细胞从皮质斑块到囊泡的形成。我们将测试LC如何调节Sla2与肌动蛋白细胞骨架相互作用的两个模型。此外，我们计划筛选与LC-NT有功能冗余的蛋白质，这将为我们提供这个系统中更完整的调控图景。在当今的美国，内源性细胞功能障碍可能导致一系列威胁公共健康的疾病，包括：高胆固醇血症、神经疾病和肿瘤发生。虽然哺乳动物LC已经得到了很好的研究，但几乎所有与该蛋白相关的功能数据都是在体外进行评估的。由于许多参与内吞作用的哺乳动物蛋白质，包括笼蛋白，在酵母中是保守的，因此利用这个模型系统是有意义的，它使我们能够在体内分析蛋白质。这项建议将研究LC对Sla2的体内调节，并提供重要的观察结果，然后将其应用于哺乳动物系统。因此，我们的研究可能会揭示有关这一重要过程的新信息，并最终帮助制定管理这些疾病的新战略。

DESCRIPTION (provided by applicant): One our laboratory's long-term goals is to understand how clathrin light chain regulates the endocytic process. This proposal is to study LC in the Saccharomyces Cerevisiae model system, which provides the ability to knockout, replace or otherwise alter genes of interest and the availability of screens for interacting molecules or intersecting pathways. It has long been known that deletion of either the heavy (HC) or light (LC) chains of clathrin in yeast caused defects in a-factor uptake and endocytic patch progression. Our lab was one of the first to recognize that LC could suppress the endocytic defects of a HC null. In addition we demonstrated that this suppression was mediated by the N-terminus (amino acids 1-143) of LC (LC-NT). Yeast two-hybrid using LC as bait identified the PIP2 binding, talin-like protein Sla2 (the yeast homologue of the mammalian endocytic adaptors Hip1/Hip1 R) as a novel binding partner and this interaction was further mapped to LC-NT. We believe that LC suppression of HC deletion involves the regulation of Sla2 by LC. As such, the main goal of this proposal is to identify how LC regulation of Sla2 promotes endocytic progression from cortical patches to vesicle formation. We will test two models for how LC regulates Sla2 interaction the actin cytoskeleton. In addition, we plan to screen for proteins which have functional redundancy with the LC-NT, which will provide us with a more complete picture of regulation in this system. Endocytic dysfunction can contribute to a diverse list of diseases which pose threats to public health in America today, including: hypercholesterolemia, neurological disorders and tumorigenesis. Although mammalian LC is well studied, nearly all functional data pertaining to this protein was assessed in vitro. Since many of the mammalian proteins involved in endocytosis, including clathrin, are conserved in yeast it makes sense to take advantage of this model system, which allows us to analyze proteins in vivo. This proposal will examine the in vivo regulation of Sla2 by LC, and provide important observations, which can then be applied to mammalian systems. As such our studies may reveal new information concerning this vital process and eventually help produce new strategies for management of these diseases.