Neo1 and phosphatidylethanolamine contribute to vacuole membrane fusion in<i>Saccharomyces cerevisiae</i>

Abstract

<i>NEO1</i> is an essential gene in budding yeast and belongs to a highly conserved subfamily of P-type ATPase genes that encode phospholipid flippases. Inactivation of temperature sensitive <i>neo1</i>^<i>ts</i> alleles produces pleiomorphic defects in the secretory and endocytic pathways, including fragmented vacuoles. A screen for multicopy suppressors of <i>neo1-2</i>^<i>ts</i> growth defects yielded <i>YPT7</i>, which encodes a Rab7 homolog involved in SNARE-dependent vacuolar fusion. <i>YPT7</i> suppressed the vacuole fragmentation phenotype of <i>neo1-2</i>, but did not suppress Golgi-associated protein trafficking defects. Neo1 localizes to Golgi and endosomal membranes and was only observed in the vacuole membrane, where Ypt7 localizes, in retromer mutants or when highly overexpressed in wild-type cells. Phosphatidylethanolamine (PE) has been implicated in Ypt7-dependent vacuolar membrane fusion <i>in vitro</i> and is a potential transport substrate of Neo1. Strains deficient in PE synthesis (<i>psd1Δ psd2Δ</i>) displayed fragmented vacuoles and the <i>neo1-2</i> fragmented vacuole phenotype was also suppressed by overexpression of <i>PSD2</i>, encoding a phosphatidylserine decarboxylase that produces PE at endosomes. In contrast, <i>neo1-2</i> was not suppressed by overexpression of <i>VPS39</i>, an effector of Ypt7 that forms a membrane contact site potentially involved in PE transfer between vacuoles and mitochondria. These results support the crucial role of PE in vacuole membrane fusion and implicate Neo1 in concentrating PE in the cytosolic leaflet of Golgi and endosomes, and ultimately the vacuole membrane.