The functional studies on the multifunctional protein Vac8p

Abstract

Cellular processes are mediated mainly by a variety of protein-protein interactions. A subset of proteins performs more than one cellular function by interacting with different partners. In this study, I investigated the multi-functional protein Vac8p (vacuole-related 8) in the budding yeast Saccharomyces cerevisiae. Vac8p is composed of 12 armadillo repeats and anchored to the vacuolar membrane via myristoylation and palmitoylation at its amino-terminal region. Vac8p is known to be involved in various cellular processes including homotypic vacuole fusion, formation of the nucleus-vacuole junction (NVJ), piecemeal microautophagy of the nucleus (PMN), cytoplasm-to-vacuole targeting (Cvt), and vacuole inheritance. In Chapter I, I described how Vac8p and Nvj1p, an outer-nuclear membrane protein, interact and mediate formation of NVJ, one of the well characterized as membrane contact sites (MCSs), which mediates physical contact between the nucleus and the vacuole. Based on X-ray crystal structures of Vac8p-Nvj1p complexes, biochemical and cell biological studies revealed the molecular mechanism underlying NVJ formation. In Chapter II, by comparing X-ray crystal structures of Vac8p-Nvj1p and Vac8p-Atg13p, a protein essential for various autophagic processes, I proposed a molecular model of how Vac8p adopts different quaternary structures depending on its associated proteins to differentially regulate 2 closely related bud distinct cellular pathways. In Chapter III, I described a molecular mechanism by which formation and disruption of NVJ is regulated. Phosphorylation of a specific amino-acid residue of Vac8p is required for formation of NVJ, and Ksp1p appears to be responsible for this phosphorylation. NVJ formation usually occurs well, but it is dirupted when vacuole inheritance proceeds to the bud during mitosis. These findings revealed that part of vacuoles have to be transported to the bud during mitosis, a portion of Vac8p is likely dephosphorylated by an unknown phosphatase prior to mitosis, and Ksp1p may re-phosphorylates Vac8p for formation of NVJ in the bud at the end of mitosis.