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dc.contributor.authorXu, Huaxi
dc.date.accessioned2018-07-12T18:39:50Z
dc.date.available2018-07-12T18:39:50Z
dc.date.issued1993
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 54-04, Section: B, page: 1761.;Advisors: Dennis Shields.
dc.identifier.urihttps://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9325663
dc.identifier.urihttps://hdl.handle.net/20.500.12202/3492
dc.description.abstractMost small peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage to generate the mature hormone. A characteristic feature of peptide hormone secreting cells is their ability to store the mature hormone in membrane-bound secretory granules. Morphological evidence from several laboratories has implicated distal Golgi/Trans-Golgi Network (TGN) as the site where prohormone cleavage may be initiated. However, little is known about the molecular mechanism whereby precursors and the mature hormones are sorted from constitutively secreted proteins in the TGN and packaged into secretory granules. To characterize the cellular machinery that mediates these processes, I established an in vitro system from retrovirally infected rat anterior pituitary GH{dollar}\sb3{dollar} cells which express high levels of prosomatostatin (proSRIF), as well as endogenous growth hormone (GH). ProSRIF could be accumulated quantitatively in the TGN by incubating the cells at 20{dollar}\sp\circ{dollar}C. Permeabilized cells were prepared and incubated in the presence of cytosol, ATP and GTP at 37{dollar}\sp\circ{dollar}C. Under these conditions, proSRIF was cleaved to the mature hormone. Prohormone cleavage required ATP but not GTP. Furthermore, proSRIF cleavage in vitro was inhibited by the non-hydrolysable ATP analogue ATP{dollar}\gamma{dollar}S or weak bases or protonophores suggesting that the function of ATP in processing is to generate a proton gradient and hence an acidic pH within the TGN for maximal prohormone processing enzyme activity. In contrast to proteolytic cleavage, budding of SRIF and GH containing vesicles from the TGN required both GTP and cytosol, in addition to ATP. Addition of the non-hydrolyzable GTP analogue GTP{dollar}\gamma{dollar}S to the system inhibited post-TGN vesicle formation but had no effect on prohormone cleavage, suggesting that GTP-binding proteins may be involved. If permeabilized cells were washed repeatedly with physiological salt, surprisingly, there appeared to be no cytosol requirement for in vitro budding. However, when permeabilized cells were treated with high salt to remove any putative budding factors, the high salt washed permeabilized cells were unable to support vesicle budding, whereas proSRIF cleavage was unaffected. Vesicle budding activity was completely restored by addition of cytosol or dialysed salt-extract to the salt-washed system. These data are consistent with our hypothesis that tightly bound budding factors are present on the surface of TGN membranes. My results suggest that prohormone cleavage occurs in the TGN prior to vesicle budding and proteolytic processing can be uncoupled from subsequent packaging of the hormones into immature secretory granules.
dc.publisherProQuest Dissertations & Theses
dc.subjectCellular biology.
dc.subjectAnimal Physiology.
dc.titleProhormone processing and packaging into nascent secretory vesicles in vitro
dc.typeDissertation


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