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.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/32635 |
| Date | 16 August 2012 |
| Creators | Tuhman-Mushkin, Jana |
| Contributors | Rubinstein, John |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.002 seconds