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。细胞壁生物合成，一个过程，需要协调的表达和运输细胞壁酶的质膜在酵母细胞中的肌球蛋白II（Myo 1 p）的缺陷显着影响。chs 3和myo 1突变之间的合成致死性和Nikkomycin Z（Chs 3 p的竞争性抑制剂）在myo 1细胞中诱导的致死性表明，myo 1细胞中的细胞壁结构严重受损。我们感兴趣的是鉴定和表征Myo 1 p依赖的运动功能（S）负责这种表型，该提案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。这项建议的目的是扩大我们的初步遗传和 生物化学研究确定了Myo 1 p在内体-TGN通路中的假定作用。为了实现这一目标，我们将进行遗传和生化分析，以确定基因，抑制尼可霉素Z诱导的myo 1菌株的致死性。通过对这些蛋白质进行功能性定量和定性检测，我们期望在涉及Myo 1 p的内体囊泡运输途径中鉴定新的蛋白质相互作用。这个建议的中心假设是，酿酒酵母的肌球蛋白II是重要的蛋白质的正常循环内体区室。我们观察到，Chs 3 p（一种通过内体再循环到质膜的完整膜蛋白）的定位受到Myo 1 p缺陷的干扰，正如Chs 3 p在点状组织中的积累所证明的那样 胞质结构和增加的Chs 3 p定位到液泡。我们预测，其他回收的蛋白质和它们的靶向受体，共享相同的回收途径，将经历定性和定量的变化，可以通过遗传筛选和其去产物的质谱分析，体内定位和生化分析来鉴定。该提议的具体目的是：1）鉴定抑制尼可霉素Z诱导的myo 1细胞致死性的基因。2)确定Chs 3 p内体转运是如何被myo 1菌株中的这些抑制基因修饰的。3)在野生型和myo 1菌株中通过比较蛋白质组学分析鉴定抑制蛋白的相互作用蛋白复合物。我们期望这项工作将导致识别 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.