Role of the v -SNARE Bet1p in cell wall biosynthesis.
Item
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Title
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Role of the v -SNARE Bet1p in cell wall biosynthesis.
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Identifier
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AAI3063845
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identifier
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3063845
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Creator
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Kipnis, Pearl.
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Contributor
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Adviser: Peter N. Lipke
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Date
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2002
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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
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Abstract
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The cell wall of Saccharomyces cerevisiae is a complex structure of cross-linked glucans, chitin and mannoproteins. The biosynthesis of the cell wall depends on the secretory pathway for components and required enzymes. To elucidate mechanisms involved in secretion and biosynthesis of the cell wall, I used W303-1B mutants previously selected for temperature sensitivity for growth and inability to cross-link the mannoprotein alpha-agglutinin to the cell wall. Twenty mutants which did not bind to a-cells were further screened for excretion of alpha-agglutinin, sensitivity to lysis by the enzyme Zymolyase, and for growth rate. Ten of these were tested for sensitivity to calcofluor white. Various strains exhibited abnormal levels of excretion of alpha-agglutinin, and all strains exhibited some degree of cell wall aberration.;To identify the genes responsible for these aberrant cell wall phenotypes I transformed the mutants with a YCp50 yeast genomic library. A mutant strain that is hyper-sensitive to Zymolyase and calcofluor white, AC59, was restored to viable growth at the restrictive temperature of 37°C by a particular plasmid from the library. The complementing gene on the plasmid proved to be BET1, which codes for a v-SNARE, an integral membrane protein on vesicles involved in ER to Golgi transport.;In AC59, incubated at the restrictive temperature, levels of invertase were diminished at the cell surface, consistent with a bet1 block in secretion, but alpha-agglutinin continued to be excreted. This apparent paradox could be explained by the existence of a post-ER pool of alpha-agglutinin.;In parent strain W303-1B the existence of a pool of alpha-agglutinin was shown by continued secretion of alpha-agglutinin to the cell wall after a cycloheximide block. In AC59 at 37°C the results were similar, except alpha-agglutinin was excreted into the media instead of being cross-linked to the cell wall.;Such a cell wall anchorage deficit could result from the absence of beta-1,6 glucan and/or enzyme activities required for anchorage. The levels of beta-1,6 glucan were assayed and found to be significantly diminished at the restrictive temperature. This work thus demonstrates a linkage between the yeast v-SNARE Bet1p, secretion and cell wall biosynthesis.
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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.
