Polarized epithelial cells efficiently sort newly-synthesized apical and basolateral proteins into distinct transport carriers that emerge from the trans-Golgi network (TGN), and this sorting may be recapitulated in nonpolarized cells. While the targeting signals of basolaterally-destined proteins are generally cytoplasmically-disposed, apical sorting signals are not typically accessible to the cytosol, and the transport machinery required for segregation and export of apical cargo remain largely unknown. We are interested in identifying the molecular requirements for TGN export of the apical marker influenza hemagglutinin (HA). To identify cytosolic proteins responsible for HA export from the TGN, we developed an in vitro assay to measure this process. We found that HA export does not require the brefeldin A-sensitive GTPase ARF1, but does depend on the GTPase dynamin 2. Furthermore, using biochemical fractionation, we identified the adaptor protein 14-3-3 epsilon as an effector of HA export from the TGN. This work 1) establishes a method to accurately observe anterograde TGN export of an apical cargo, 2) characterizes a requirement for GTP in vesicle formation, and 3) identifies a novel component in trafficking from the TGN. We expand these studies by comparing HA export to the release of basolateral cargo, showing that nonpolarized cells are capable of differentially sorting distinct classes of cargo into discrete vesicles derived from the TGN. Together, these data further our understanding of the regulatory mechanisms underlying apical transmembrane protein export from the TGN.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04112006-162436 |
| Date | 27 April 2006 |
| Creators | Ellis, Mark A |
| Contributors | Linton M. Traub, PhD, Jeffrey Brodsky, PhD, Meir Aridor, PhD, Ora A. Weisz, PhD, Gerard Apodaca, PhD, Rebecca P. Hughey, PhD |
| 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-04112006-162436/ |
| 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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