Assessing Function and Identifying Modifiers of Protein Isoaspartyl Methyltransferase in Schizosaccharomyces pombe

Abstract

Protein isoaspartyl methyltransferase (PIMT), an ancient enzyme with putative homologs in virtually all eukaryotes studied to date, is a protein repair enzyme that initiates restoration of age-damaged isoaspartyl residues back into normal amino acids. I studied the functional importance of PIMT in Schizosaccharomyces pombe, whose PIMT-encoding homolog is designated pcm2. Past studies of PIMT in other organisms had suggested that Pcm2p function could be important for viability under stress conditions. In the first part of the project, I compared the viability of a yeast strain lacking pcm2 under a variety of stress conditions, including stationary phase, heat shock, high-salt, high-sugar, high-pH, and high temperature. Based on studies of PIMT in other organisms, I expected to see reduced viability of the knockout strain during stress conditions, but no difference in cell growth or viability was observed. I also attempted to use Real-Time PCR to compare pcm2 transcription in yeast under the same stress conditions. I have not yet collected any usable data from the Real-Time PCR runs, but it has been previously shown that pcm2 transcription increases during stationary phase, heat shock, and osmotic shock. This increase in pcm2 transcription during stress with no phenotype associated with loss of pcm2 under stress conditions leads to the hypothesis that there may be a second gene whose product can compensate for loss of PIMT function. To locate and identify this gene, a synthetic lethal screen is being utilized. Currently, five potential synthetic lethal mutants have been isolated.