title: Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells.
abstract: Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448-19457). This study capitalized on the mechanisms suppressing vacuolar H(+)-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, 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, School of Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131.
authors.forename: Dennis
authors.initials: D
authors.lastname: Dominguez
authors.affiliation: From the Department of Biochemistry and Molecular Biology, 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: D000431:Ethanol / Q000378:metabolism; D005947:Glucose / Q000235:genetics / Q000378:metabolism; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D010732:Phosphofructokinase-1 / Q000172:deficiency*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D000431:Ethanol; D010732:Phosphofructokinase-1; D025262:Vacuolar Proton-Translocating ATPases; D005947:Glucose
keywords: glucose; glycolysis; proton pump; proton transport; vacuolar ATPase; vacuolar acidification; yeast