Type-IV P-type ATPases are putative phospholipid flippases that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. However, flippase activity has not been reconstituted with any purified type-IV P-type ATPase, and so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. The major goal of this dissertation project is to test whether Drs2p, a type-IV P-type ATPase from yeast Saccharomyces cerevisiae, is a flippase or not. I show that Drs2p can directly catalyze phospholipid translocation through purification and reconstitution of this ATPase into artificial membranes. This flippase activity is specific to a phosphatidylserine analogue and requires active Drs2p. My data also suggest that only a portion of Drs2p molecules are active after purification and these active molecules may result from partial proteolysis within the carboxyl terminus of Drs2p. This observation is consistent with a model that the carboxyl tail is auto-inhibitory to Drs2p activity. In this study, I demonstrate for the first time the reconstitution of flippase activity with a purified type-IV P-type ATPase.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-03192010-142021 |
| Date | 19 March 2010 |
| Creators | Zhou, Xiaoming |
| Contributors | Todd R. Graham, Hassane S. Mchaourab, Charles R. Sanders, Katherine L. Friedman, Carl H. Johnson |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-03192010-142021/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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