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.
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