title: Neo1 and phosphatidylethanolamine contribute to vacuole membrane fusion inSaccharomyces cerevisiae.
abstract: NEO1is 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 sensitiveneo1tsalleles produces pleiomorphic defects in the secretory and endocytic pathways, including fragmented vacuoles. A screen for multicopy suppressors ofneo1-2tsgrowth defects yieldedYPT7, which encodes a Rab7 homolog involved in SNARE-dependent vacuolar fusion.YPT7suppressed the vacuole fragmentation phenotype ofneo1-2, 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 fusionin vitroand is a potential transport substrate of Neo1. Strains deficient in PE synthesis (psd1Δ psd2Δ) displayed fragmented vacuoles and theneo1-2fragmented vacuole phenotype was also suppressed by overexpression ofPSD2, encoding a phosphatidylserine decarboxylase that produces PE at endosomes. In contrast,neo1-2was not suppressed by overexpression ofVPS39, 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.
authors.affiliation: Department of Biological Sciences, Vanderbilt University , Nashville, TN, USA.
authors.forename: Yuantai
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Biological Sciences, Vanderbilt University , Nashville, TN, USA.
authors.forename: Mehmet
authors.initials: M
authors.lastname: Takar
authors.affiliation: Department of Biological Sciences, Vanderbilt University , Nashville, TN, USA.
authors.forename: Andrea A
authors.initials: AA
authors.lastname: Cuentas-Condori
authors.affiliation: Department of Biological Sciences, Vanderbilt University , Nashville, TN, USA.
authors.forename: Todd R
authors.initials: TR
authors.lastname: Graham
keywords: P4-ATPase; Rab protein; SNARE; Ypt7; flippase; membrane asymmetry; vacuoles