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Regulation of apical vesicle formation from the trans-Golgi network

Ellis, Mark A (2006) Regulation of apical vesicle formation from the trans-Golgi network. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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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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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Ellis, Mark
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairHughey, Rebecca Phughey@dom.pitt.eduHUGHEYR
Committee MemberApodaca, Gerardgla6@pitt.eduGLA6
Committee MemberBrodsky, Jeffreyjbrodsky@pitt.eduJBRODSKY
Committee MemberTraub, Linton Mtraub@pitt.eduTRAUB
Committee MemberAridor, Meiraridor@pitt.eduARIDOR
Committee MemberWeisz, Ora Aweisz@pitt.eduWEISZ
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:, etd-04112006-162436
Date Deposited: 10 Nov 2011 19:35
Last Modified: 15 Nov 2016 13:39


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