Functional Analysis of DUF1 in Saccharomyces cerevesiae

Abstract

The yeast DUF1 gene encodes a highly‐conserved, WD40‐repeat protein that interacts with and activates deubiquitinases (DUBs). DUBs carry out the process of deubiquitination, the removal of ubiquitin from tagged proteins. Animal homologues of DUF1 have been shown to form complexes with DUBs that have a variety of important functions. Human WDR48 activates DUBs that deubiquitinate key subunits in the Fanconi anemia DNA repair pathway. Caenorhabditis elegans WDR48 interacts with Usp46 and stabilizes Glutamate receptors (GLR‐1) to promote changes in locomotion behavior. Previous research has shown that in Saccharomyces cerevisiae, Duf1 interacts with Ubp9 and Ubp13, DUBs involved in the biosynthesis of mitochondrial subunits necessary for respiration. When a DUF1 mutant strain was grown at a higher temperature on a non‐fermentable carbon source, increased petite colony formation was observed. Thus, we hypothesize that Duf1 enhances the yeast's ability to cope with stress. Our interest lies in determining which cellular stress pathways require Duf1 in order to gain insight about its function. By exposing a DUF1 knockout mutant strain to different stress conditions and comparing its growth to the wild type parent strain, phenotypes may be revealed to provide a better understanding of the cellular activities of Duf1. Cell growth experiments were performed in 96‐well plates using a Spectramax i3 plate reader and cell number was plotted against time. Growth rate and maximum cell number were computed in R and differences between the knockout mutant and the wild type parent strain were determined using a pairwise Mann‐Whitney U test. When grown in minimal media, the knockout mutant strain grew faster than the wild type parent strain. The knockout mutant was also able to reach a larger maximum cell number than that of the wild type parent strain in minimal media containing high levels of glucose, a stressful growth situation. Conversely, the wild type parent strain grew better when minimal media was supplemented with high amounts of ethanol. This implies that the action of Duf1 as either a positive or negative regulator is dependent upon the type of stress experienced and that DUF1 may play a role in multiple cellular stress response pathways.Support or Funding InformationThis project is supported by an NSF Major Research Instrumentation grant and Salisbury University's Henson Undergraduate Research Fund.