Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human

Abstract

Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys 79 in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys 79 methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated. Molecular & Cellular Proteomics

MS2‐Deisotoper: A Tool for Deisotoping High‐Resolution MS/MS Spectra in Normal and Heavy Isotope‐Labelled Samples

“…The methods used for preparing yeast samples for MS/MS have been described elsewhere . Briefly, cells were lysed, protein extracts were separated by SDS‐PAGE, and gel bands prepared for mass spectrometry analysis.…”

The ribosome: A hot spot for the identification of new types of protein methyltransferases

“…In human EF1A, the N-terminal glycine also appears to be trimethylated, although the adjacent lysine residue appears to be unmodified (69). The methyltransferase responsible for this modification has not been identified.…”

“…However, Efm2 has been shown to recognize lysine residues on both EF2 and EF3 (5). A perhaps bigger surprise was revealed with the characterization of yeast Efm7 that appears to catalyze the modification of both the Nterminal α-amino group and the side chain ε-amino group of Lys-2 of yeast elongation factor 1A (69). Efm7 may be the first example of a protein methyltransferase that specifically recognizes one protein substrate but then catalyzes methylation reactions at different types of residues within that substrate.…”

Chemical Biology of Protein N‐Terminal Methyltransferases

“…In addition to α‐N‐terminal methylation on the classical X‐P‐K/R motif in eukaryotic cells, a novel N‐terminal methylation has been recently reported on eukaryotic elongation factor 1A (eEF1A) in both yeast and humans . YLR285W, also named elongation factor methyltransferase 7 (Efm7), is a dual MTase that installs methyl groups at both N‐terminal Gly1 and Lys2 residues of yeast eEF1A protein . Lys2 is methylated only after trimethylation of Gly1 .…”

“…Recently, human MTase‐like protein 13 (METTL13) was identified as a dual MTase for both N‐terminal Gly1 and Lys55 of human eEF1A . Human eEF1A contains a very similar N‐terminal sequence, GKEKTHIN, with Thr substituted at the fifth position instead of Ser, as seen in yeast . METTL13 has two distinct MTase domains: N‐ and C‐terminal domains that appear to have different recognition preferences.…”