Protein glutaminylation is a yeast-specific posttranslational modification of elongation factor 1A

Abstract

Ribosomal translation factors are fundamental for protein synthesis and highly conserved in all kingdoms of life. The essential eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl tRNAs to the A-site of the translating 80S ribosome. Several studies have revealed that eEF1A is posttranslationally modified. Using MS analysis, site-directed mutagenesis, and X-ray structural data analysis of Saccharomyces cerevisiae eEF1A, we identified a posttranslational modification in which the α amino group of mono-l-glutamine is covalently linked to the side chain of glutamate 45 in eEF1A. The MS analysis suggested that all eEF1A molecules are modified by this glutaminylation and that this posttranslational modification occurs at all stages of yeast growth. The mutational studies revealed that this glutaminylation is not essential for the normal functions of eEF1A in S. cerevisiae However, eEF1A glutaminylation slightly reduced growth under antibiotic-induced translational stress conditions. Moreover, we identified the same posttranslational modification in eEF1A from Schizosaccharomyces pombe but not in various other eukaryotic organisms tested despite strict conservation of the Glu⁴⁵ residue among these organisms. We therefore conclude that eEF1A glutaminylation is a yeast-specific posttranslational modification that appears to influence protein translation.

Keywords: GTPase; Saccharomyces cerevisiae; Schizosaccharomyces pombe; eukaryotic elongation factor eEF1A; glutaminylation; helix A*–loop–helix A′ region; posttranslational modification (PTM); protein synthesis; switch I region; translation elongation factor.

Figure 1.

eEF1A isolated from S. cerevisiae is posttranslationally modified in the GTPase domain at Glu⁴⁵. A, extracted ion chromatogram showing that the tryptic peptide 42-FEKEAAELGK-51 ([M+2H]²⁺ = 625.3251) of yeast EF1A is covalently modified by the attachment of a mass of 128.059 Da. B, MS-MS fragmentation analysis revealed that the tryptic peptide 42-FEKEAAELGK-51 of yeast EF1A is modified at a side chain of Glu⁴⁵. Immon., immonium ion; Seq., sequence.

Figure 2.

Confirmation of eEF1A modification by site-directed mutagenesis of Glu⁴⁵ in S. cerevisiae. A, MS-MS fragmentation analysis by collision-induced dissociation (CID) of the eEF1A E45D mutant expressed and purified from S. cerevisiae genetically deprived of wild-type eEF1A. A representative MS-MS fragmentation pattern (left panel) and extracted ion chromatogram (right panel) are shown. Posttranslational modification of the tryptic peptide 42-FEKDAAELGK-51 of eEF1A was not observed. B, electron transfer dissociation (ETD) MS-MS analysis of the eEF1A E45A mutant expressed and purified from S. cerevisiae genetically deprived of wild-type eEF1A. Posttranslational modification of the chymotryptic peptide 43-EKAAAELGKGSFKY-56 was not observed. A representative MS-MS fragmentation pattern (left panel) and extracted ion chromatogram (right panel) are shown.

Figure 3.

Crystal structure of glutaminylated yeast eEF1A. A, the crystal structure of posttranslationally modified eEF1A from the eEF1A–eEF1B complex. The helix A*–loop–helix A′ region of the GTPase domain (brown) of eEF1A is marked. The modified Glu⁴⁵ is shown as sticks and balls. Domain II and domain III are shown in cyan and green, respectively. eEF1B was omitted for clarity. Original structure factors were deduced from Ref. . B, electron density map 2mF_o–dF_c (gray), contoured at a level of 1.7 σ in the vicinity of the l-glutaminyl moiety (yellow) covalently attached via its α amino group to Glu⁴⁵ of yeast eEF1A. C, chemical structure of the covalent linkage of the glutaminyl moiety (red) to Glu⁴⁵.

Figure 4.

Glu⁴⁵ in translation elongation factor 1A is highly conserved from archaea to human. Shown is the amino acid sequence alignment of the N terminus of eEF1A of various organisms. The alignment was prepared with ClustalW with eEF1A from S. cerevisiae (accession no. P02994), S. pombe (accession no. P0CT53), M. musculus (accession no. P10126), B. taurus (accession no. P68103), G. mellonella (accession no. Q8MWN6), D. rerio (accession no. Q92005), Homo sapiens (accession no. P68104), and H. volcanii (accession no. D4GWR0). Secondary structural elements were deduced from S. cerevisiae eEF1A (PDB code 1IJF). The N-terminal part of the switch I region, which is missing in prokaryotic EF-Tu, is highlighted.

Figure 5.

Mouse elongation factor 1A is not glutaminylated at Glu⁴⁵. A, SDS-PAGE of eEF1A affinity-purified from mouse liver (mEF1A), which was used for mass spectrometric analysis. B, CID MS-MS revealed no modification of Glu⁴⁵ in the chymotryptic peptide 43-EKEFAAEMGKGSF-54 (precursor m/z = 642.3 (2+)). Immon., immonium ion; Seq., sequence. C, a representative LC/MS-MS extracted ion chromatogram of the peptide 43-EKEAAEMGKGSF-54 peak at m/z = 642.3 (2+) from mouse eEF1A.

Figure 6.

Glutaminylation or mutation of eEF1A Glu⁴⁵ to lysine increased growth defect under translational stress conditions in yeast. Growth phenotypes of S. cerevisiae strains expressing wild-type eEF1A Glu⁴⁵-glut, eEF1A E45D, or eEF1A E45K. Serial dilutions of the corresponding S. cerevisiae variants were spotted onto YPGal (2% galactose) or YPD agar supplemented with different stress agents (50 ng/ml Geneticin, 350 μg/ml paromomycin (Paro), or 5 μg/ml anisomycin (Aniso)) and cultivated for 72 h at 30 °C.