Intracellular trafficking and degradation of unassociated proalpha 2 chains of collagen type I.
Item
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Title
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Intracellular trafficking and degradation of unassociated proalpha 2 chains of collagen type I.
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Identifier
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AAI9924813
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identifier
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9924813
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Creator
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Gotkin, Marilyn Gallup.
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Contributor
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Adviser: Robert S. Bienkowski
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Date
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1999
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell
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Abstract
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Background. Procollagen (I) is a trimer consisting of two proalpha I chains and one proalpha 2 chain. In mild Osteogenesis Imperfecta (O.I.), abnormal proalpha 1 chains are degraded in the endoplasmic reticulum. As a consequence, the cells produce excess proalpha 2 chains, which cannot form trimers and are not secreted.;Objective. The objective of this work was to determine the intracellular fate of unassociated proalpha 2 chains.;Methods. This study used Mov13 cells, which do not synthesize proalpha 1 mRNA or chains, but do produce mRNA for proalpha 2 chains. Cells were incubated with radioactive isotopes using pulse-chase protocols. In some experiments, cells were exposed to brefeldin-A or wortmannin to perturb the secretory pathway. Proteins were analyzed by gel electrophoresis, autoradiography, Western blotting and immunoprecipitation, Cells were imaged by confocal laser scanning microscopy using markers for proalpha 2, Golgi and lysosomes.;Results. Mov13 cells produced proalpha 2 chains that were quickly degraded and were not secreted. Degradation was inhibited when cells were treated with 500 ng brefeldin-A, which collapses the Golgi into the endoplasmic reticulum and inhibits the secretion of newly synthesized proteins. Immunofluorescence studies showed that proalpha 2 chains colocalize with markers for Golgi and lysosomes. Treatment with 2 muM wortmannin, which inhibits trafficking to lysosomes, caused proalpha 2 chains to be secreted.;Conclusion. These results are taken as evidence that unassociated proalpha 2 chains can exit the endoplasmic reticulum, transit the Golgi and enter lysosomes where they are degraded.;Significance. This investigation is important because it has yielded insights into how cells react to the pathological conditions characteristic of O.I., and it can further our general understanding of how cells process excess subunits of heteromeric proteins.
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Type
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dissertation
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Source
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PQT Legacy Restricted.xlsx
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degree
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Ph.D.