The yeast vacuole is a dynamic organelle that is functionally analogous to the mammalian lysosome and serves as a model for the study of membrane fusion and fission. Mechanisms of membrane fission and fusion dynamics have been well conserved from yeast to humans. However, the regulatory mechanisms that govern cellular fission and fusion dynamics remain poorly understood. Our lab has previously established that Env7 is a conserved yeast palmitoylated protein kinase that localizes to the yeast vacuole and negatively regulates vacuole membrane fusion during budding and hyperosmotic stress. Phosphorylation of Env7 is dependent on another vacuolar membrane kinase, Yck3, and is essential to Env7 stability and negative regulation of vacuolar membrane fusion. In this study, we aim to further our understanding of the role Env7 plays at the vacuole by 1) characterizing the phosphorylation of Env7 as a function of cell cycle using cell cycle arrest and synchronization techniques, and 2) generating functional biochemically tagged Yck3 to be used in interaction and phosphorylation assays with Env7. Cell cycle arrest and synchronization techniques have not previously been established in our lab. Here, we report reliable protocols of inducing cell cycle arrest using α-factor mating pheromone and Hydroxyurea. Results show that Env7 is hyperphosphorylated when cell cycle is arrested at G1 phase using α-factor mating pheromone. In both cell cycle arrest approaches, vacuoles show significant increase in fragmentation, and Env7 remains localized to the membrane of fragmented vacuoles. In cell culture synchronized with α-factor, Env7 shows an increase in phosphorylation between S-phase and G2, with decreased phosphorylation in M and G1. We were successful in engineering biochemically tagged Yck3 and established that the expressed 6XHis-Yck3 is functional and able to restore phosphorylation of Env7 in vivo. We also established that overexpressed 6XHis-Yck3 localized correctly to the vacuolar membrane. These tools will be used in future studies on interactions and regulation of membrane fusion.
|Commitee:||Bhandari, Deepali, Fraser, Deborah|
|School:||California State University, Long Beach|
|School Location:||United States -- California|
|Source:||MAI 58/01M(E), Masters Abstracts International|
|Subjects:||Biology, Molecular biology, Cellular biology|
|Keywords:||Env7, Lysosome, Membrane biogenesis, Phosphorylation, Vacuole fission/fusion, Yeast|
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