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Post-translational modification by ?-lysylation is required for activity of Escherichia coli elongation factor P (EF-P).

ABSTRACT: Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical ?-lysyl-lysine. Here we present biochemical evidence for ?-lysyl-lysine modification in Escherichia coli EF-P and for its role in EF-P activity by characterizing native and recombinant EF-P proteins for their modification status and activity in vitro. Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native and recombinant EF-P proteins. The ?-lysyl-lysine isopeptide was identified in the exhaustive Pronase digests of native EF-P and recombinant EF-P isolated from E. coli coexpressing EF-P, YjeA, and YjeK but not in the digests of proteins derived from the vectors encoding EF-P alone or EF-P together with YjeA, indicating that both enzymes, YjeA and YjeK, are required for ?-lysylation of EF-P. Endogenous EF-P as well as the recombinant EF-P preparation containing ?-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis ?4-fold over the preparations containing unmodified EF-P and/or ?-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the ?-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.

SUBMITTER: Park JH

PROVIDER: S-EPMC3268417 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Post-translational modification by β-lysylation is required for activity of Escherichia coli elongation factor P (EF-P).

Park Jong-Hwan JH Johansson Hans E HE Aoki Hiroyuki H Huang Bill X BX Kim Hee-Yong HY Ganoza M Clelia MC Park Myung Hee MH

The Journal of biological chemistry 20111129 4

Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical β-lysyl-lysine ...[more]

PMID: 22128152

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Project description:UnlabelledElongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of wild-type and efp mutant Salmonella. We found that metabolic and motility genes are prominent among the subset of proteins with decreased production in the Δefp mutant. Furthermore, particular tripeptide motifs are statistically overrepresented among the proteins downregulated in efp mutant strains. These include both PPP and PPG but also additional motifs, such as APP and YIRYIR, which were confirmed to induce EF-P dependence by a translational fusion assay. Notably, we found that many proteins containing polyproline motifs are not misregulated in an EF-P-deficient background, suggesting that the factors that govern EF-P-mediated regulation are complex. Finally, we analyzed the specific region of the PoxB protein that is modulated by EF-P and found that mutation of any residue within a specific GSCGPG sequence eliminates the requirement for EF-P. This work expands the known repertoire of EF-P target motifs and implicates factors beyond polyproline motifs that are required for EF-P-mediated regulation.ImportanceBacterial cells regulate gene expression at several points during and after transcription. During protein synthesis, for example, factors can interact with the ribosome to influence the production of specific proteins. Bacterial elongation factor P (EF-P) is a protein that facilitates the synthesis of proteins that contain polyproline motifs by preventing the ribosome from stalling. Bacterial cells that lack EF-P are viable but are sensitive to a large number of stress conditions. In this study, a global analysis of protein synthesis revealed that EF-P regulates many more proteins in the cell than predicted based solely on the prevalence of polyproline motifs. Several new EF-P-regulated motifs were uncovered, thereby providing a more complete picture of how this critical factor influences the cell's response to stress at the level of protein synthesis.

Project description:BackgroundThe bacterial elongation factor P (EF-P) is strictly conserved in bacteria and essential for protein synthesis. It is homologous to the eukaryotic translation initiation factor 5A (eIF5A). A highly conserved eIF5A lysine is modified into an unusual amino acid derived from spermidine, hypusine. Hypusine is absolutely required for eIF5A's role in translation in Saccharomyces cerevisiae. The homologous lysine of EF-P is also modified to a spermidine derivative in Escherichia coli. However, the biosynthesis pathway of this modification in the bacterial EF-P is yet to be elucidated.Presentation of the hypothesisHere we propose a potential mechanism for the post-translational modification of EF-P. By using comparative genomic methods based on physical clustering and phylogenetic pattern analysis, we identified two protein families of unknown function, encoded by yjeA and yjeK genes in E. coli, as candidates for this missing pathway. Based on the analysis of the structural and biochemical properties of both protein families, we propose two potential mechanisms for the modification of EF-P.Testing the hypothesisThis hypothesis could be tested genetically by constructing a bacterial strain with a tagged efp gene. The tag would allow the purification of EF-P by affinity chromatography and the analysis of the purified protein by mass spectrometry. yjeA or yjeK could then be deleted in the efp tagged strain and the EF-P protein purified from each mutant analyzed by mass spectrometry for the presence or the absence of the modification. This hypothesis can also be tested by purifying the different components (YjeK, YjeA and EF-P) and reconstituting the pathway in vitro.Implication of the hypothesisThe requirement for a fully modified EF-P for protein synthesis in certain bacteria implies the presence of specific post-translational modification mechanism in these organisms. All of the 725 bacterial genomes analyzed, possess an efp gene but only 200 (28%) possess both yjeA and yjeK genes. In the other organisms, EF-P may be modified by another pathway or the translation machinery must have adapted to the lack of EF-P modification. Our hypotheses, if confirmed, will lead to the discovery of a new post-translational modification pathway.ReviewersThis article was reviewed by Céline Brochier-Armanet, Igor B. Zhulin and Mikhail Gelfand. For the full reviews, please go to the Reviewers' reports section.

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Post-translational modification by ?-lysylation is required for activity of Escherichia coli elongation factor P (EF-P).

Publications

Post-translational modification by β-lysylation is required for activity of Escherichia coli elongation factor P (EF-P).

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