The regulation of glucose starvation-induced cell surface protein clearance in yeast

 描述(由申请人提供)：正确的细胞内膜运输确保跨膜、分泌和细胞器腔内驻留的“货物”蛋白质在细胞内正确定位。膜运输对于正常的细胞生理是必不可少的。它通过控制细胞的信号传递能力、粘附性和内化营养物质的能力来调节细胞的增殖和行为。交通方面的缺陷与癌症和 应激状态下的细胞死亡。膜交通及其调控是一种非常保守的古老过程。由于其易用性，酿酒酵母为研究这种保守的途径提供了一个理想的模型系统。最近，邓肯实验室描述了在葡萄糖饥饿时，酵母细胞内化了许多细胞表面蛋白，并在液泡中降解它们。这一过程对细胞生存至关重要；然而，调节这种膜运输变化的机制尚不清楚。使用一种候选方法，我发现了一种蛋白质，它对葡萄糖饥饿过程中蛋白质的内化和降解非常重要。在这项提案中，我将测试候选蛋白质在酵母中以葡萄糖依赖的方式进行调节的模型，以推动细胞表面蛋白质的降解，并最终导致细胞存活。我的初步数据显示，在葡萄糖饥饿期间，候选蛋白质减少了货物蛋白向液泡的运输，但在葡萄糖存在的情况下不会。这些数据与候选者在内吞作用或在细胞内隔间分选中的作用是一致的。因此，我将首先使用上位性分析结合生物化学和荧光显微镜来确定候选蛋白质在葡萄糖饥饿期间作用于哪一膜。下一步，我将确定这种蛋白质在葡萄糖饥饿期间如何调节的分子机制。我将测试候选蛋白质是否受到翻译后修饰，或者它与结合伙伴的相互作用是否在葡萄糖饥饿期间发生变化。为此，我将应用免疫印迹分析或GST下拉和免疫沉淀与质谱学相结合的方法来检测和识别葡萄糖饥饿期间翻译后修饰和结合蛋白的变化。然后，我将通过突变影响修饰或相互作用来测试翻译后修饰或结合伙伴的功能意义。这些研究将为了解葡萄糖饥饿引起的膜运输变化的调控机制提供必要的数据。了解这些调控机制将使我们能够了解酵母细胞如何在葡萄糖饥饿中幸存下来，并提供一个框架来测试癌细胞或其他人类细胞在与疾病相关的应激条件下是否使用同样的机制。

 DESCRIPTION (provided by applicant): Correct intracellular membrane traffic ensures that transmembrane, secreted and organelle lumen resident "cargo" proteins are correctly positioned within the cell. Membrane traffic is essential for normal cell physiology. It regulates cell proliferation and behavior by controlling the signaling capacity of cells, their adhesive propertie, and their capacity to internalize nutrients. Defects in traffic are associated with cancer and with cell death during stress. Membrane traffic and its regulation are ancient processes that are highly conserved. Due to its ease of use, the yeast Saccharomyces cerevisiae provides an ideal model system for the study of such conserved pathways. Recently, the Duncan lab described that upon glucose starvation, yeast cells internalize many cell surface proteins and degrade them in the vacuole. This process is essential for cell survival; however, the mechanism that regulates this change in membrane traffic is unknown. Using a candidate approach, I discovered one protein that is important for the internalization and degradation of proteins during glucose starvation. In this proposal, I will test the model that the candidate protein is regulated in a glucose-dependent manner in yeast to drive cell surface protein degradation and ultimately cell survival. My preliminary data shows that the candidate protein reduces the delivery of cargo proteins to the vacuole during glucose starvation but not when glucose is present. The data are consistent with a role for the candidate in endocytosis or in sorting at intracellular compartments. Thus, I will first determine at which membrane the candidate protein acts during glucose starvation using epistasis analysis coupled with biochemistry and fluorescence microscopy. Next, I will determine the molecular mechanism for how this protein is regulated during glucose starvation. I will test if the candidate protein is subject to post-translational modification or if its interactions with binding partners change during glucose starvation. To do this, I will apply approaches such as immuno-blot analysis or GST-pull-down and immuno-precipitation coupled with mass spectrometry to detect and identify changes in post-translational modification and binding proteins during glucose starvation. I will then test th functional significance of the post-translational modification or binding partner by making mutations that affect the modification or interaction. These studies will provide the necessary data to understand the mechanism regulating glucose starvation induced changes in membrane traffic. Understanding these regulatory mechanisms will allow us to understand how yeast cells survive glucose starvation and provide a frame-work to test if this same mechanism is used by cancer cells or other human cells under stress conditions related to disease.