Methylation of proteins occurs predominantly on arginine and lysine residues in the eukaryotic cell. Until recently, its predominance was unknown and its role obscure. This presentation outlines our efforts to construct the first ‘methylproteome network’ for a eukaryotic cell and presents evidence that methylation modulates protein-protein interactions in this network.
We analysed the yeast methylproteome to identify methylated proteins and precise modification sites. Targeted data acquisition - electron transfer dissociation LC-MS/MS was used, as were yeast proteome arrays (containing 4,400 chips spotted on to microscope slides). To build the intracellular methylation network, all known and putative methyltransferases in yeast were knocked out and the methylproteome re-analysed to determine which enzyme was responsible for which methylation event. Enzyme-substrate links were further investigated by the analysis of recombinant substrate proteins methylated by recombinant enzymes, by in vivo methylation assays and/or the incubation of proteome arrays with recombinant enzymes. Validated enzyme-substrate links were integrated with yeast protein-protein interactions to generate the first ‘methylproteome network’. A new ‘conditional two-hybrid' (C2H) system was then constructed to test whether methylation can modulate protein-protein interactions.
Our analyses, together, showed that protein methylation is widespread in the eukaryotic cell. We discovered two new eukaryotic lysine methyltransferases, elongation factor methyltransferases 2 and 3, both of which have mammalian orthologs. Our integrated methyltransferase-substrate protein and protein-protein interaction network suggested that methylation might modulate protein-protein interactions. This was proven by or new 'conditional two hybrid' system, in that half of the protein-protein pairs involving arginine methylated proteins show a significant increase in interaction on methylation. In some cases, phosphorylation was found adjacent to methylation and we have shown that this can interfere with methylation and thus decrease certain protein-protein interactions.
The 'methylproteome network', built here, reveals a novel means of regulating protein-protein interactions and thus biological function in the eukaryotic cell.