Clathrin-mediated endocytosis is the major process by which cells internalize nutrients, extracellular macromolecules, and membrane constituents to regulate such diverse processes as cell polarity, development, and motility. Over twenty proteins comprise a large protein interaction web that is pertinent to this process. This work investigates proteins that act as clathrin-associated sorting proteins (CLASPs), and their interactions with other endocytic components. Epsin 1 is shown to be a CLASP that engages components of the endocytic clathrin coat and selects for polyubiquitinated cargo. The interaction with polyubiquitin is enabled through epsin 1s UIM domains. I show that polyubiquitin is an efficient endocytic signal, which is relevant for physiological mammalian substrates such as the epithelial sodium channel (ENaC). Stonin 2 behaves as an unconventional CLASP, as it doesnt directly engage clathrin or the plasma membrane. My work uses this protein to biochemically characterize the WXXF motif and identify a privileged binding site located on the sandwich subdomain of the AP-2 ? appendage. This work supports a model in which arrays of binding motifs and multiple engagement sites on the ? appendage allow for an increase in binding affinity, which affects the temporal ordering of endocytic accessory protein interactions during clathrin-mediated endocytosis. These studies have defined important protein interactions that have improved our understanding of the molecular mechanism of clathrin-mediated endocytosis.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08272006-230458 |
| Date | 30 August 2006 |
| Creators | Hawryluk, Matthew John |
| Contributors | Edwin S. Levitan, Ph.D., John P. Johnson, M.D., Gerard L. Apodaca, Ph.D., Meir Aridor, Ph.D. |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-08272006-230458/ |
| 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 University of Pittsburgh 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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