Identification of Methylated Mitochondrial Proteins in Saccharomyces cerevisiae

Protein methylation has recently emerged as an abundant and important posttranslational modification, affecting many cellular functions including transcription and translation. The budding yeast Saccharomyces cerevisiae has become an important model organism for studying methylation reactions and the enzymes responsible for catalyzing the reaction (methyltransferases). Generally, either unfractionated lysates from cells grown under fermentation or specific protein substrates of well-characterized methyltransferases have been investigated for protein methylation. As a result of this, the methylation state of proteins inside the mitochondria remains largely unknown. The goal of this dissertation was two-fold: 1.) determine the experimental requirements for thoroughly identifying the methylation state of proteins in yeast mitochondria and 2.) identify methylated proteins in mitochondria that may have been previously overlooked. Only one example in the scientific literature has investigated the global level of protein methylation in the mitochondria of any organism and only two proteins in the yeast mitochondria are known to be methylated. I set out to determine mitochondrial protein methylation by detecting differences between wild type and methyltransferase gene deletion yeast strains using a combination of commonly used biochemical and molecular biology assays. When I found that these methods were ill-suited for identification of mitochondrial protein methylation, I turned to mass spectrometry approaches. From my initial mass spectrometric analyses of proteolytic digests of yeast proteins, I learned that it was not sufficient to simply rely on computer algorithms to identify novel methylated peptides. It is important to use isotopic labeling to validate the mass shifts that occur as a result of methylation on identified peptides. This led me to use to adapt heavy methyl stable isotope labeling with amino acids in cell culture (SILAC) for use in yeast with my identification of a strain better suited for these experiments. From this approach, I have been able to curate a list of proteins comprising the mitochondrial methyl proteome. Using this method, I have also compared the cytosolic methyl proteome between yeast cells grown under fermentative or respiratory conditions. I conclude with future directions for the work included in this dissertation. The functional implications of the mitochondrial methylation reactions have yet to be discovered and the methyltransferases responsible remain to be identified. This dissertation contains three supplemental Excel spreadsheets. These materials are available on ProQuest.com. The first supplemental Excel table is an expanded version of Table 2-1 with a list of the known and putative methyltransferases in yeast, their confirmed and predicted substrate type, the substrate and residue methylated if known, and descriptions of the methyltransferase. The second Excel table contains data from nine separate mass spectrometry experiments, attempting to identify the methylation state of mitochondrial ribosomal proteins from wild type and methyltransferase gene deletion strains, including peptides identified and descriptions. The third supplemental Excel table contains the peptides identified as methylated in cytosol isolated from fermenting and respiring yeast cells