Investigation of the role of yeast BiP in secretory pathway function and regulation.
Item
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Title
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Investigation of the role of yeast BiP in secretory pathway function and regulation.
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Identifier
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AAI9405581
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identifier
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9405581
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Creator
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Rocco, James William.
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Contributor
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Adviser: Reza Green
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Date
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1993
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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, Molecular | Biology, Cell | Biology, General
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Abstract
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In this study we examined the role of yeast BiP, a luminal ER heat shock protein, in the maturation of a triple glycosylation mutant of yeast prepro-{dollar}\alpha{dollar}-factor (N123-pp{dollar}\alpha{dollar}f). Using pulse-chase radiolabeling in combination with SDS-PAGE and HPLC, we initially observed that the mutant strain exhibited a severe defect in the secretion of {dollar}\alpha{dollar}f compared to wild-type cells. Surprisingly, treatment of mutant cells with tunicamycin (TM), an inhibitor of Asn-linked glycosylation, enhanced the transport and processing of N123-pp{dollar}\alpha{dollar}f, suggesting a possible trans-acting involvement of yeast BiP. By constructing recombinant yeast strains in which BiP expression was independent of TM treatment, and under the control of the heterologous CUP1 (metallothionein) promoter we were able to independently regulate the level of intracellular BiP. We observed that overexpression of BiP led to the retention, and subsequent degradation, of N123-p{dollar}\alpha{dollar}f in a post-ER compartment. This suggested that, rather than promoting folding and transport, BiP plays a negative role in the lumen of the ER by binding mutant and malfolded proteins and promoting their degradation. Support for an interaction between BiP and N123-p{dollar}\alpha{dollar}f was supported by two additional results: First, expression of N123-p{dollar}\alpha{dollar}f resulted in a dose-dependent transcriptional induction of BiP mRNA. Second, co-expression of BiP and N123-p{dollar}\alpha{dollar}f was inhibitory to growth in a dose-dependent manner.;Using these same recombinant strains, we also demonstrated that overexpression of BiP from the heterologous CUP1 promoter, prior to challenging the yeast cells with TM, protected the cells from the cytotoxic action of the drug, in analogy to the protective role of cytoplasmic heat shock proteins in response to thermal stress. To our knowledge, this is the first example that BiP plays a role in protecting the cell from a lethal ER stress.;Finally, the TM-mediated transcriptional induction of BiP can be suppressed by heterologous overexpression of BiP from the CUP1 promoter, suggesting that BiP is directly involved in regulating its own transcription.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
