title: Reversible disassembly of the yeast V-ATPase revisited under in vivo conditions.
abstract: Primary active proton transport by eukaryotic V-ATPases (vacuolar ATPases) is regulated via the reversible disassembly of the V1Vo holoenzyme into its peripheral catalytic V1 complex and its membrane-bound proton-translocating Vo complex. This nutrient-dependent phenomenon had been first detected in the midgut epithelium of non-feeding moulting tobacco hornworms (Manduca sexta) and in glucose-deprived yeast cells (Saccharomyces cerevisiae). Since reversible disassembly to date had been investigated mostly in vitro, we wanted to test this phenomenon under in vivo conditions. We used living yeast cells with V-ATPase subunits fused to green, yellow or cyan fluorescent protein and found that only the V1 subunit C (Vma5) was released into the cytosol after substitution of extracellular glucose with galactose, whereas the other V1 subunits remained at or near the membrane. FRET analysis demonstrated close proximity between V1 and Vo even under glucose-starvation conditions. Disassembly, but not reassembly, depended on functional microtubules. Results from overlay blots, pull-down assays and bimolecular fluorescence complementation support the assumption that subunit C interacts directly with microtubules without involvement of linker proteins.
authors.affiliation: *Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Tabke
authors.affiliation: *Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Albertmelcher
authors.affiliation: *Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Vitavska
authors.affiliation: *Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Huss
authors.affiliation: †Department of Biology/Chemistry, Division of Genetics, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Schmitz
authors.affiliation: *Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Wieczorek
mesh_terms: D003600:Cytosol / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases