Reversible disassembly of the yeast V-ATPase revisited under in vivo conditions

Autor(en): Tabke, Katharina 
Albertmelcher, Andrea
Vitavska, Olga 
Huss, Markus 
Schmitz, Hans-Peter 
Wieczorek, Helmut 
Stichwörter: Biochemistry & Molecular Biology; CELLS; DISRUPTION; DISSOCIATION; GLUCOSE; HIGH-EFFICIENCY TRANSFORMATION; MEMBRANE; microtubule; PROTEINS; reversible disassembly; Saccharomyces cerevisiae; SACCHAROMYCES-CEREVISIAE; SUBUNIT-C; vacuolar ATPase (V-ATPase); VACUOLAR H+-ATPASE
Erscheinungsdatum: 2014
Herausgeber: PORTLAND PRESS LTD
Journal: BIOCHEMICAL JOURNAL
Volumen: 462
Ausgabe: 1
Startseite: 185
Seitenende: 197
Zusammenfassung: 
Primary active proton transport by eukaryotic V-ATPases (vacuolar ATPases) is regulated via the reversible disassembly of the V1V0 holoenzyme into its peripheral catalytic V-1 complex and its membrane-bound proton-translocating V-0 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 V-1 subunit C (Vma5) was released into the cytosol after substitution of extracellular glucose with galactose, whereas the other Vi subunits remained at or near the membrane. FRET analysis demonstrated close proximity between V-1 and V-0 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.
ISSN: 02646021
DOI: 10.1042/BJ20131293

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