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相关。细胞壁生物合成是酵母细胞中需要细胞壁酶协调表达和转运至质膜的过程，该过程受到肌球蛋白 II (Myo1p) 缺陷的显着影响。 chs3 和 myo1 突变之间的综合致死性以及 Chs3p 竞争性抑制剂 Nikkomycin Z 在 myo1 细胞中诱导的致死性表明 myo1 细胞的细胞壁结构受到严重损害。我们对导致这种表型的 Myo1p 依赖性运动功能的识别和表征感兴趣。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。该提案的目的是扩大我们初步遗传和 定义 Myo1p 在内体到 TGN 途径中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 Nikkomycin Z 诱导致死的基因。通过对这些蛋白质进行功能定量和定性检查，我们期望识别涉及 Myo1p 的内体囊泡运输途径中新的蛋白质相互作用。该提议的中心假设是酿酒酵母的肌球蛋白 II 对于内体区室中蛋白质的正常回收很重要。我们观察到 Chs3p（一种完整的膜蛋白，通过内体再循环到质膜）的定位受到 Myo1p 缺陷的干扰，Chs3p 在点状细胞中的积累证明了这一点。 细胞质结构和 Chs3p 在液泡中的定位增加。我们预测，共享相同回收途径的其他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选来识别，并通过对其消失产物的质谱分析、体内定位和生化分析进行表征。该提案的具体目标是：1）鉴定抑制 Nikkomycin Z 诱导的 myo1 细胞致死的基因。 2)确定myo1菌株中这些抑制基因如何改变Chs3p内体运输。 3) 通过野生型和 myo1 菌株的比较蛋白质组分析来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之间的新联系 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.