Methylation has emerged as an essential modification of small molecules, lipids, nucleic acids, and proteins. Given the variety of potential substrates, the effects the addition of a small methyl group can have are expansive and diverse. Methylation of DNA can turn a gene off and methylation of a tRNA can affect its stability. Proteins offer even more variety of methylatable targets; methyl groups can be added to the side chains of lysine, arginine, glutamine, glutamate, asparagine, histidine, methionine, and cysteine as well as the N- and C-terminal amino and carboxyl groups of alanine, serine, proline, phenylalanine, and leucine. The most well-known function of protein methylation is arguably the modification of histone tails, part of the histone code that regulates gene expression. However, there are a number of non-histone methylations that are often less well understood in terms of functional relevance. Interestingly, in Saccharomyces cerevisiae, a large number of methylation reactions occur on translation-related proteins including ribosomal proteins, release factors, and elongation factors. This fact served as the initial spark for a variety of my projects that centered on identifying the enzymes responsible for these reactions and delving into potential roles of the methylation reactions.
The elongation factors in S. cerevisiae are heavily methylated containing a total of ten methylated lysine residues. The original focus of this dissertation was to identify as many of the enzymes for these ten reactions as possible. Two had been identified at the onset of this project and this dissertation adds two more along with some evidence for a third. In my work, Efm3 was shown to trimethylate lysine 509 on elongation factor 2 and Efm5 was shown to catalyze the trimethylation of lysine 79 of elongation factor 1A. I also obtained preliminary evidence that YNL024C catalyzes the monomethylation of lysine 390 on EF1A. This identification was later confirmed by another group and the enzymes was designated Efm6. This second group also suggested that there is a relatively low occupancy of this modification, which explains the difficulty we had confirming the activity with our methods.
An interesting discovery was made during this search for elongation factor methyltransferases: a potentially new type of protein methylation that has yet to be described. It is possible that this methylation went undetected in previous screens and analyses due to its unusual characteristics and its behavior in our assays. I have been able to show that a radioactive product from acid hydrolysates of yeast cells labeled with S-adenosyl-[methyl-3H]-L-methionine is only formed in cells expressing the YLR285W gene. This gene encodes a putative methyltransferase but has not been associated directly with a substrate in vivo. This dissertation describes my attempts made to identify this new substrate and modification but as of yet, it remains elusive.
