Genetic and Proteomic Analysis of Myosin II-Dependent Endosome Transport

肌球蛋白II传统上与胞质分裂和细胞分裂的功能有关。在酵母细胞中，细胞壁生物发生是一个需要细胞壁酶的协调表达和向质膜运输的过程，它受到肌球蛋白II(Myo1p)缺乏的显著影响。Chs3和myo1突变之间的合成致死性以及由chs3p的竞争性抑制剂nikmycin Z诱导的myo1细胞的致死性表明，myo1细胞的细胞壁结构严重受损。我们感兴趣的是与这种表型相关的Myo1p依赖的运动功能(S)的鉴定和特征，这一提议的长期目标是了解肌球蛋白II在真核细胞生长和形态发生中的功能。这项建议的目标是扩大我们初步的遗传和 确定Myo1p在内体到TGN途径中的假定作用的生化研究。为了实现这一目标，我们将进行遗传和生化分析，以确定在myo1菌株中抑制尼克霉素Z诱导的致死性的基因。通过对这些蛋白质进行功能定量和定性研究，我们期望在内体囊泡运输途径(S)中发现涉及Myo1p的新的蛋白质相互作用。这一提议的中心假设是酿酒酵母的肌球蛋白II对于内吞体内蛋白质的正常循环是重要的。我们观察到，Chs3p(一种完整的膜蛋白，通过内吞体内再循环到质膜)的定位受到Myo1p缺乏的干扰，这一点就是Chs3p积累的证据。 细胞质结构和Chs3p在液泡中的定位增加。我们预测，其他具有相同回收途径的回收蛋白及其靶向受体将经历定性和定量的变化，这些变化可以通过基因筛查来识别，并通过对其消失的产物进行质谱分析、体内定位和生化分析来表征。这项建议的具体目的是：1)识别抑制尼克霉素Z诱导的myo1细胞致死性的基因。2)确定这些抑制基因在myo1菌株中是如何改变Chs3p内体转运的。3)通过比较蛋白质组学分析鉴定野生型和myo1菌株中抑制蛋白的相互作用蛋白复合体。我们期望，这项工作将有助于确定 肌球蛋白II和参与内体囊泡运输网络的蛋白质。

Myosin II is traditionally associated with functions in cytokinesis and cell division. Cell wall biogenesis, a process that requires coordinated expression and transport of cell wall enzymes to the plasma membrane in yeast cells is significantly affected by myosin II (Myo1p) deficiency. Synthetic lethality between chs3 and myo1 mutations and lethality induced in myo1 cells by Nikkomycin Z, a competitive inhibitor of Chs3p, suggests that cell wall architecture is severely compromised in myo1 cells. We are interested in the identification and characterization of Myo1p-dependent motility function(s) responsible for this phenotype, The long-range goal of this proposal is to understand the function of myosin II in eukaryotic cell growth and morphogenesis. The objective of this proposal is to expand the results of our preliminary genetic and biochemical studies that define a putative role for Myo1p in the endosome-to-TGN pathway. To achieve this objective we will conduct a genetic and biochemical analysis to identify genes that suppress Nikkomycin Z-induced lethality in myo1 strains. By conducting functional quantitative and qualitative examination of these proteins, we expect to identify new protein interactions in the endosome vesicle traffic pathway(s) that involve Myo1p. The central hypothesis of this proposal is that myosin II of Saccharomyces cerevisiae is important for the normal recycling of proteins in the endosome compartment. We have observed that the localization of Chs3p (an integral membrane protein that is recycled to the plasma membrane through the endosome) is perturbed by Myo1p deficiency, as evidenced by the accumulation of Chs3p in punctate cytoplasmic structures and increased localization of Chs3p to the vacuole. We predict that other recycled proteins and their targeting receptors that share the same recycling pathway, will undergo qualitative and quantitative changes that can be identified by genetic screens and characterized by mass spectrometry analysis of their gone products, in vivo localization and biochemical analysis. The specific aims of this proposal are to: 1) identify genes that suppress Nikkomycin Z-induced lethality in myo1 cells. 2) Determine how Chs3p endosomal transport is modified by these suppressor genes in myo1 strains. 3) identify interacting protein complexes of suppressor proteins by a comparative proteomic analysis in wild type and myo1 strains. It is our expectation that this work will lead to the identification of novel connections between myosin II and proteins involved in the endosome vesicle transport network.