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Structural Studies of Saccharomyces cerevisiae V1-ATPase in the Stationary Phase of Yeast Cell Culture

Vacuolar-type ATPases (V-ATPases) are ubiquitous membrane-bound protein complexes present in the endo-membrane system of all eukaryotic cells. In eukaryotic cells, the reversible dissociation of the V1 and Vo regions is an essential mechanism for regulating V-ATPase activity. Therefore, knowledge of the structure of the dissociated V1-ATPase is necessary for understanding the regulation of V-ATPase activity. In this thesis, I showed that by introducing a 3xFLAG tag at the C terminus of different V1-ATPase subunits, highly purified V1-ATPase complex could be isolated. Electron cryomicroscopy (cryo-EM) was used for initial analysis of the intact V1-ATPase. In addition to the intact complex, partial V1-ATPase subcomplexes with different subunit compositions were isolated from yeast cells in late log phase. All of the isolated subcomplexes were found to contain the major V1-ATPase subunits A and B, but differed in the peripheral stalk subunit composition.

Identiferoai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32635
Date16 August 2012
CreatorsTuhman-Mushkin, Jana
ContributorsRubinstein, John
Source SetsUniversity of Toronto
Languageen_ca
Detected LanguageEnglish
TypeThesis

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