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.
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Research Article|
July 24 2014
Reversible disassembly of the yeast V-ATPase revisited under in vivo conditions
Katharina Tabke;
Katharina Tabke
1
*Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany
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Andrea Albertmelcher;
Andrea Albertmelcher
1
*Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany
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Olga Vitavska;
Olga Vitavska
*Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany
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Markus Huss;
Markus Huss
*Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany
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Hans-Peter Schmitz;
Hans-Peter Schmitz
†Department of Biology/Chemistry, Division of Genetics, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany
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Helmut Wieczorek
Helmut Wieczorek
2
*Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastrasse 11, 49069 Osnabrück, Germany
2To whom correspondence should be addressed (email wieczorek@biologie.uni-osnabrueck.de).
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Publisher: Portland Press Ltd
Received:
November 21 2013
Revision Received:
April 10 2014
Accepted:
May 07 2014
Accepted Manuscript online:
May 07 2014
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2014 Biochemical Society
2014
Biochem J (2014) 462 (1): 185–197.
Article history
Received:
November 21 2013
Revision Received:
April 10 2014
Accepted:
May 07 2014
Accepted Manuscript online:
May 07 2014
Citation
Katharina Tabke, Andrea Albertmelcher, Olga Vitavska, Markus Huss, Hans-Peter Schmitz, Helmut Wieczorek; Reversible disassembly of the yeast V-ATPase revisited under in vivo conditions. Biochem J 15 August 2014; 462 (1): 185–197. doi: https://doi.org/10.1042/BJ20131293
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