Aug 25, 2015

Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting

BioRxiv : the Preprint Server for Biology
David MavorJames Fraser

Abstract

Ubiquitination is an essential post-translational regulatory process that can control protein stability, localization, and activity. Ubiquitin is essential for eukaryotic life and is highly conserved, varying in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies in S. cerevisiae indicate that ubiquitin is highly tolerant to single amino acid mutations. To resolve this paradox, we hypothesized that the set of tolerated substitutions would be reduced when the cultures are not grown in rich media conditions and that chemically induced physiologic perturbations might unmask constraints on the ubiquitin sequence. To test this hypothesis, a class of first year UCSF graduate students employed a deep mutational scanning procedure to determine the fitness landscape of a library of all possible single amino acid mutations of ubiquitin in the presence of one of five small molecule perturbations: MG132, Dithiothreitol (DTT), Hydroxyurea (HU), Caffeine, and DMSO. Our data reveal that the number of tolerated substitutions is greatly reduced by DTT, HU, or Caffeine, and that these perturbations uncover “shared sensitized positions” localized to areas around the hydrophobic patch and to the C-terminus...Continue Reading

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Mentioned in this Paper

Establishment and Maintenance of Localization
Study
DRUG Screen Quant Caffeine
Proteasome Pathway
Dimethyl Sulfoxide
Regulation of Biological Process
Ubiquitin
Determination Aspects
Yeasts
Dithiothreitol

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