title: Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly.
abstract: V-ATPases are conserved ATP-driven proton pumps that acidify organelles. Yeast V-ATPase assembly and activity are glucose-dependent. Glucose depletion causes V-ATPase disassembly and its inactivation. Glucose readdition triggers reassembly and resumes proton transport and organelle acidification. We investigated the roles of the yeast phosphofructokinase-1 subunits Pfk1p and Pfk2p for V-ATPase function. The pfk1Δ and pfk2Δ mutants grew on glucose and assembled wild-type levels of V-ATPase pumps at the membrane. Both phosphofructokinase-1 subunits co-immunoprecipitated with V-ATPase in wild-type cells; upon deletion of one subunit, the other subunit retained binding to V-ATPase. The pfk2Δ cells exhibited a partial vma growth phenotype. In vitro ATP hydrolysis and proton transport were reduced by 35% in pfk2Δ membrane fractions; they were normal in pfk1Δ. In vivo, the pfk1Δ and pfk2Δ vacuoles were alkalinized and the cytosol acidified, suggestive of impaired V-ATPase proton transport. Overall the pH alterations were more dramatic in pfk2Δ than pfk1Δ at steady state and after readdition of glucose to glucose-deprived cells. Glucose-dependent reassembly was 50% reduced in pfk2Δ, and the vacuolar lumen was not acidified after reassembly. RAVE-assisted glucose-dependent reassembly and/or glucose signals were disturbed in pfk2Δ. Binding of disassembled V-ATPase (V1 domain) to its assembly factor RAVE (subunit Rav1p) was 5-fold enhanced, indicating that Pfk2p is necessary for V-ATPase regulation by glucose. Because Pfk1p and Pfk2p are necessary for V-ATPase proton transport at the vacuole in vivo, a role for glycolysis at regulating V-ATPase proton transport is discussed.
authors.affiliation: From the Department of Biochemistry and Molecular Biology of the School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
authors.forename: Chun-Yuan
authors.initials: CY
authors.lastname: Chan
authors.affiliation: From the Department of Biochemistry and Molecular Biology of the School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131 kjparra@salud.unm.edu.
authors.forename: Karlett J
authors.initials: KJ
authors.lastname: Parra
mesh_terms: D005947:Glucose / Q000235:genetics / Q000378:metabolism*; D006019:Glycolysis / Q000502:physiology; D007425:Intracellular Membranes / Q000201:enzymology; D025481:Phosphofructokinase-2 / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology / Q000235:genetics
chemical_list: D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D025481:Phosphofructokinase-2; D025262:Vacuolar Proton-Translocating ATPases; D005947:Glucose
keywords: Glucose; Phosphofructokinase; RAVE; Saccharomyces cerevisiae; V-ATPase; V1Vo-ATPase; Vacuolar ATPase; Vacuolar Acidification; Yeast