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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 Glu 45 residue among these organisms. We therefore conclude that eEF1A glutaminylation is a yeast-specific posttranslational modification that appears to influence protein translation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Legionella is a pathogenic Gram-negative bacterium that can multiply inside of eukaryotic cells. It translocates numerous bacterial effector proteins into target cells to transform host phagocytes into a niche for replication. One effector of Legionella pneumophila is the glucosyltransferase Lgt1, which modifies serine 53 in mammalian elongation factor 1A (eEF1A), resulting in inhibition of protein synthesis and cell death. Here, we demonstrate that similar to mammalian cells, Lgt1 was severely toxic when produced in yeast and effectively inhibited in vitro protein synthesis. Saccharomyces cerevisiae strains, which were deleted of endogenous eEF1A but harbored a mutant eEF1A not glucosylated by Lgt1, were resistant toward the bacterial effector. In contrast, deletion of Hbs1, which is also an in vitro substrate of the glucosyltransferase, did not influence the toxic effects of Lgt1. Serial mutagenesis in yeast showed that Phe(54), Tyr(56) and Trp(58), located immediately downstream of serine 53 of eEF1A, are essential for the function of the elongation factor. Replacement of serine 53 by glutamic acid, mimicking phosphorylation, produced a non-functional eEF1A, which failed to support growth of S. cerevisiae. Our data indicate that Lgt1-induced lethal effect in yeast depends solely on eEF1A. The region of eEF1A encompassing serine 53 plays a critical role in functioning of the elongation factor.

Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt's), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNA(Phe) and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNA(His) and Phe-tRNA(Phe)) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt's and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt's., (© 2011 Tzivelekidis et al.)