PT  - JOURNAL ARTICLE
AU  - David Mavor
AU  - Kyle A. Barlow
AU  - Samuel Thompson
AU  - Benjamin A. Barad
AU  - Alain R. Bonny
AU  - Clinton L. Cario
AU  - Garrett Gaskins
AU  - Zairan Liu
AU  - Laura Deming
AU  - Seth D. Axen
AU  - Elena Caceres
AU  - Weilin Chen
AU  - Adolfo Cuesta
AU  - Rachel Gate
AU  - Evan M. Green
AU  - Kaitlin R. Hulce
AU  - Weiyue Ji
AU  - Lillian R. Kenner
AU  - Bruk Mensa
AU  - Leanna S. Morinishi
AU  - Steven M. Moss
AU  - Marco Mravic
AU  - Ryan K. Muir
AU  - Stefan Niekamp
AU  - Chimno I. Nnadi
AU  - Eugene Palovcak
AU  - Erin M. Poss
AU  - Tyler D. Ross
AU  - Eugenia Salcedo
AU  - Stephanie See
AU  - Meena Subramaniam
AU  - Allison W. Wong
AU  - Jennifer Li
AU  - Kurt S. Thorn
AU  - Shane Ó. Conchúir
AU  - Benjamin P. Roscoe
AU  - Eric D. Chow
AU  - Joseph L. DeRisi
AU  - Tanja Kortemme
AU  - Daniel N. Bolon
AU  - James S. Fraser
TI  - Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting
AID  - 10.1101/025452
DP  - 2015 Jan 01
TA  - bioRxiv
PG  - 025452
4099  - http://biorxiv.org/content/early/2015/08/25/025452.short
4100  - http://biorxiv.org/content/early/2015/08/25/025452.full
AB  - 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. We also show perturbation specific effects including the sensitization of His68 in HU and tolerance to mutation at Lys63 in DTT. Taken together, our data suggest that chemical stress reduces buffering effects in the ubiquitin proteasome system, revealing previously hidden fitness defects. By expanding the set of chemical perturbations assayed, potentially by other classroom-based experiences, we will be able to further address the apparent dichotomy between the extreme sequence conservation and the experimentally observed mutational tolerance of ubiquitin. Finally, this study demonstrates the realized potential of a project lab-based interdisciplinary graduate curriculum.