Ellis, Mark A
(2006)
Regulation of apical vesicle formation from the trans-Golgi network.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
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.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
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| ETD Committee: |
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| Date: |
27 April 2006 |
| Date Type: |
Completion |
| Defense Date: |
23 January 2006 |
| Approval Date: |
27 April 2006 |
| Submission Date: |
11 April 2006 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Cell Biology and Molecular Physiology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
apical; biosynthetic trafficking; HA; in vitro assay; TGN; TGN export |
| Other ID: |
http://etd.library.pitt.edu/ETD/available/etd-04112006-162436/, etd-04112006-162436 |
| Date Deposited: |
10 Nov 2011 19:35 |
| Last Modified: |
15 Nov 2016 13:39 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/6996 |
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