RT Journal Article
SR Electronic
T1 Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 025452
DO 10.1101/025452
A1 David Mavor
A1 Kyle A. Barlow
A1 Samuel Thompson
A1 Benjamin A. Barad
A1 Alain R. Bonny
A1 Clinton L. Cario
A1 Garrett Gaskins
A1 Zairan Liu
A1 Laura Deming
A1 Seth D. Axen
A1 Elena Caceres
A1 Weilin Chen
A1 Adolfo Cuesta
A1 Rachel Gate
A1 Evan M. Green
A1 Kaitlin R. Hulce
A1 Weiyue Ji
A1 Lillian R. Kenner
A1 Bruk Mensa
A1 Leanna S. Morinishi
A1 Steven M. Moss
A1 Marco Mravic
A1 Ryan K. Muir
A1 Stefan Niekamp
A1 Chimno I. Nnadi
A1 Eugene Palovcak
A1 Erin M. Poss
A1 Tyler D. Ross
A1 Eugenia Salcedo
A1 Stephanie See
A1 Meena Subramaniam
A1 Allison W. Wong
A1 Jennifer Li
A1 Kurt S. Thorn
A1 Shane Ó. Conchúir
A1 Benjamin P. Roscoe
A1 Eric D. Chow
A1 Joseph L. DeRisi
A1 Tanja Kortemme
A1 Daniel N. Bolon
A1 James S. Fraser
YR 2015
UL http://biorxiv.org/content/early/2015/08/25/025452.abstract
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.