1 research outputs found
Lysine methylation in the interactome of Saccharomyces cerevisiae
Protein methylation is emerging as an important regulator of protein-protein interactions. This thesis examined lysine methylation, a modification whose extent and significance is poorly understood, in the interactome of Saccharomyces cerevisiae. The predominance of methyllysine was explored through protein-specific enrichment utilising a combinatorial hexapeptide ligand library. This approach reduced the dynamic range of protein abundance in whole cell lysate of S. cerevisiae. Precursor ion scanning and tandem mass spectrometry (MS/MS), in association with antibody-based detection, was then used to identify lysine-methylated proteins and the precise sites of modification. This identified 20 novel methylated lysine residues. Methyltransferases for these sites were screened using single knockout strains of known and putative methyltransferases. Methylated residues associated with specific methyltransferases were identified and confirmed on peptides using high resolution MS/MS. Our experiments revealed that elongation factor 1-α is mono-methylated by Efm1 at lysine 30 and di-methylated by See1 at lysine 316. Methyltransferase Rkm1 was found to mono-methylate 40S ribosomal protein S18-A/B at lysine 48. Knockout analysis also revealed that putative methyltransferase YBR271W affects the methylation of elongation factor protein 2 and 3A. We proposed that enzyme YBR271W be renamed Efm2 (elongation factor methyltransferase 2), in line with the recent naming of YHL039W as Efm1. To elucidate the possible role of lysine methylation in the regulation of protein-protein interactions, we employed stable isotope labelling by amino acids in cell culture (SILAC). We also developed a novel approach incorporating SILAC with native-PAGE electrophoresis to monitor changes in protein complexes. Our findings showed lysine methylation to be of multifunctional purpose in the cell. Single gene knockouts of methyltransferases resulted in the change in abundance of methylated and unmethylated proteins, in some protein complexes and highlighted a possible role of methylation in the process of protein translation