Development of a Saccharomyces cerevisiae protein stability assay and its use to identify a new Clb2 degradation motif [electronic resource].

Hendrickson, Carrie Anne.
184 p.
Contained In:
Dissertation Abstracts International 63-02B.

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Molecular biology.
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The identification of ubiquitin-mediated proteolysis (UMP) substrates and of the mechanism of their polyubiquitination is important to understanding how UMP regulates cell cycle progression. To study the mechanism of substrate selection, a colorimetric assay using expression of Ade2p to complement the red ade colony phenotype was developed for the visualization of protein levels in the yeast Saccharomyces cerevisiae. Fusion protein levels are visualized as differences in yeast colony color and prototrophy: stable fusion proteins produce white ADE colonies and unstable fusions produce red ade colonies. Yeast transformants expressing Ade2p fused to the UMP-target proteins Clb2p and Pds1p form red colonies and do not accumulate the fusion protein whereas those expressing Ade2p fused to the stable protein GFP form white colonies and do accumulate Ade2p. Direct comparison of yeast colony color and fusion protein level shows strong correlation and suggests that colony color is indicative of protein stability. Mutation of the Clb2p Destruction box only partially stabilizes the protein. In addition, Clb2p sequence analysis revealed two potential KEN motifs, a recently identified degradation signal. We used our Ade2 color assay, in combination with Western analysis, to study the effect of KEN85 or KEN100 on the stability of a Clb2p N-terminal 124 amino acid fragment. We found that levels of both wildtype Clb2p and a KEN85 mutant were low but levels of a KEN100 mutant were elevated. Measurement of full length Clb2p degradation rates confirmed the increased stability of the KEN100 mutant in G1. Upon mutation of both the Destruction box and KEN100, Clb2p was further stabilized during M-A and G1. These results indicate that both degradation motifs are necessary to properly regulate Clb2p proteolysis from the initiation of anaphase through G1. Thus, this thesis advances the current understanding of how a specific substrate, Clb2p, is targeted for UMP in a cell cycle dependent manner, and provides a visual assay for the rapid identification of novel degradation motifs in substrates of targeted proteolysis.
Source: Dissertation Abstracts International, Volume: 63-02, Section: B, page: 0666.
Adviser: Sandra Holloway.
Thesis (Ph.D.)--University of Pennsylvania, 2002.
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School code: 0175.
University of Pennsylvania.
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