Uncovering the Roles of EAF1 in the Regulation of Lipid Synthesis and Membrane Composition in Saccharomyces Cerevisiae

Abstract

The yeast lysine acetyltransferase NuA4 has been implicated in regulating various aspects of metabolism, including a poorly defined role in lipid homeostasis. While classically known to acetylate histones, regulating gene expression, NuA4 also targets several non-histone proteins for acetylation. Here, I use a combination of functional genomics and fluorescent microscopy to define a novel role for NuA4 in regulating phospholipid availability for organelle morphology. First, I characterized that disruption of the NuA4 complex resulted in 70% of cells displaying deformed nuclei and nearly 50% of cells exhibiting high levels of vacuolar fragmentation. Interestingly, the deformed nuclei appeared to occur at all stages of the cell cycle, indicating an underlying dysregulation of lipids driving phospholipid metabolism. With deletion or mutation of additional genes involved in phospholipid synthesis, I was able to partially rescue nuclear deformation but not vacuolar fragmentation. To investigate the cause of these phenotypes further, I probed whether there could be a defect in a critical autophagy process known as piecemeal microautophagy of the nucleus (PMN). PMN acts to selectively degrade excess nuclear membrane into the vacuole and is performed at the nuclear-vacuole junction (NVJ). Interestingly, cells deficient in NuA4 also showed severe defects in the formation of NVJ and the function of PMN. To determine the cause of these defects, I sought to determine if these phenotypes were due to an uncharacterized relationship between NuA4 and Pah1, an enzyme that converts phosphatidic acid into diacylglycerol, which favours the accumulation of lipid droplets over phospholipids that are used for membrane expansion. NuA4 subunit Eaf1 was required for Pah1 localization to the inner nuclear membrane, and artificial tethering of Pah1 to the nuclear membrane rescued nuclear flare and vacuole fragmentation defects but not defects related to the formation of NVJs. Mutation of a candidate NuA4 acetylation site on Pah1 also resulted in aberrant Pah1 localization and defects in membrane morphology and NVJ. This work defines a critical role for NuA4 modification of Pah1 in the regulation of lipid pools that define the characteristics and function of organelle membranes. Given the high level of conservation of NuA4 with the human Tip60 complex, as well as Pah1 with human lipin, this work is highly relevant to human disease.