SNAP-25 and syntaxin 4 are SNARE proteins that are involved in membrane transport. In order for proteins to traffic properly through membranous organelles, a series of budding and fusion events must occur between donor and acceptor membranes. Therefore, determining the precise complex of proteins that are responsible for these events within the cell is critical in understanding this fundamental cellular process. In this document, I show that cytLEK1, a relatively large protein that contains numerous leucine zippers, directly binds both SNAP-25 and syntaxin 4. Through this association identified by a yeast two-hybrid screen, the protein complex regulates plasma membrane trafficking. I identified the binding domain within each of the proteins that is responsible for interaction, and performed co-immunoprecipitation and colocalization studies to confirm their association. Further analyses show that VAMP2, also a member of the SNARE complex, in contained within the cytLEK1-SNAP-25-syntaxin 4 complex. Using cytLEK1 dominant negative and knock-down approaches, I show that cytLEK1 functions in two processes that regulate the recycling endosome network: transferrin and GLUT4-trafficking. Previous work has shown that cytLEK1 interacts with the microtubule cytoskeleton. We postulate that cytLEK1 links recycling endosomes with the microtubule network. This is the first report linking these two subcelluar systems. I have also created a conditional Lek1 knock-out mouse line. By utilizing a mouse line that expresses heart specific Cre, I am able to examine Lek1 loss-of-function during heart organogensis. Through my pilot studies, I am able to show that both myocardial wall structure and function are severely altered in conditional Lek1 knock-out mice. My data show that the phenotypes may be due to the inability of cardiomyocytes to traffic proteins properly, therefore altering cell coupling and overall heart function. Taken together, my studies show that cytLEK1 is an integral member of the plasma membrane recycling pathway, and cytLEK1 function is critical in heart development.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-11302006-131209 |
| Date | 04 December 2006 |
| Creators | Pooley, Ryan Dee |
| Contributors | Steve Hann, David Bader, Susan Wente, H. Scott Baldwin, Maureen Gannon |
| 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-11302006-131209/ |
| 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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