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Methylation of translation‐associated proteins in S accharomyces cerevisiae : Identification of methylated lysines and their methyltransferases
Author(s) -
Couttas Timothy A.,
Raftery Mark J.,
Padula Matthew P.,
Herbert Ben R.,
Wilkins Marc R.
Publication year - 2012
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100570
Subject(s) - methyltransferase , methylation , protein methylation , biochemistry , lysine , biology , ribosomal protein , microbiology and biotechnology , o methyltransferase , gene , amino acid , ribosome , rna
This study aimed to identify sites of lysine methylation in S accharomyces cerevisiae and the associated methyltransferases. Hexapeptide ligand affinity chromatography was used to normalize the abundance levels of proteins in whole cell lysate. MS / MS , in association with antibody‐based detection, was then used to identify lysine methylated proteins and the precise sites of modification. Lysine methylation was found on the proteins elongation factor ( EF ) 1‐α, 2, and 3 A , as well as ribosomal proteins 40 S S 18‐ A / B , 60 S L 11‐ A / B , L 18‐ A / B , and L 42‐ A / B . Precise sites were mapped in all cases. Single‐gene knockouts of known and putative methyltransferase(s), in association with MS / MS , showed that EF 1‐α is monomethylated by E fm1 at lysin 30 and dimethylated by S ee1 at lysine 316. Methyltransferase R km1 was found to monomethylate 40 S ribosomal protein S 18‐ A / B at lysine 48. Knockout analysis also revealed that putative methyltransferase YBR 271 W affects the methylation of proteins EF 2 and 3 A ; this was detected by Western blotting and immunodetection. This methyltransferase shows strong interspecies conservation and a tryptophan‐containing motif associated with its active site. We suggest that enzyme YBR 271 W is named EF methyltransferase 2 ( E fm2), in line with the recent naming of YHL 039 W as E fm1.