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Distinct XPPX Sequence Motifs Induce Ribosome Stalling, Which Is Rescued by the Translation Elongation Factor EF-P

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Distinct XPPX Sequence Motifs Induce Ribosome Stalling, Which Is Rescued by the Translation Elongation Factor EF-P Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P Lauri Peila,b, Agata L. Starostac,d, Jürgen Lassakd,e, Gemma C. Atkinsonb, Kai Virumäef, Michaela Spitzera, Tanel Tensonb, Kirsten Jungd,e, Jaanus Remmef,1, and Daniel N. Wilsonc,d,1 aWellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom; bInstitute of Technology, University of Tartu, Tartu 50411, Estonia; cGene Center, Department for Biochemistry, Ludwig-Maximilians-Universität München, 81377 Munich, Germany; dCenter for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, 81377 Munich, Germany; eDepartment of Biology I, Microbiology, Ludwig-Maximilians- Universität München, 82152 Martinsried, Germany; and fInstitute of Molecular and Cell Biology, University of Tartu, Tartu 51010, Estonia Edited by Harry F. Noller, University of California, Santa Cruz, CA, and approved August 5, 2013 (received for review June 4, 2013) Ribosomes are the protein synthesizing factories of the cell, poly- at PPP, PPG as well as PPD and PPE triplets, despite the profiling merizing polypeptide chains from their constituent amino acids. being performed with wild-type cells containing EF-P or eIF-5A However, distinct combinations of amino acids, such as polyproline (7, 14, 15). stretches, cannot be efficiently polymerized by ribosomes, leading To address the role of EF-P during translation in E. coli to translational stalling. The stalled ribosomes are rescued by in vivo, we used SILAC (stable isotope labeling by amino acids the translational elongation factor P (EF-P), which by stimulating in cell culture) coupled with high-resolution mass spectrometry peptide-bond formation allows translation to resume. Using meta- (MS) to monitor the changes in expression levels of proteins bolic stable isotope labeling and mass spectrometry, we demon- in E. coli strains lacking either the efp gene, or one of the genes strate in vivo that EF-P is important for expression of not only (yjeK, yjeA, yfcM) encoding the modification enzymes, relative polyproline-containing proteins, but also for specific subsets of to the parental wild-type E. coli strain. We found that in the proteins containing diprolyl motifs (XPP/PPX). Together with absence of EF-P, YjeA, or YjeK, the majority of PPP-containing asystematicinvitroandinvivoanalysis,weprovideadistinct proteins are strongly down-regulated, whereas only specificsubsets hierarchy of stalling triplets, ranging from strong stallers, such as of XPP- and PPX-containing proteins are down-regulated. A sys- PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of tematic analysis of each of the 39 XPP/PPX combinations (where X BIOCHEMISTRY which are substrates for EF-P. These findings provide mechanistic means any amino acid) reveals the hierarchy of EF-P dependence. insight into how the characteristics of the specific amino acid Moreover, we show in vitro and in vivo that the combinations of substrates influence the fundamentals of peptide bond formation. strong XPP with strong PPX motifs lead to XPPX quadruplets with the strongest effects, which are nevertheless efficiently relieved rotein synthesis in the cell occurs on macromolecular ma- by EF-P. Collectively, our findings broaden the substrate range Pchines called ribosomes. The ribosome synthesizes polypeptide for EF-P activity from ∼100 PPP-containing proteins in E. coli chains by providing a platform upon which peptide-bond forma- to encompass the >1,300 additional XPPX-containing proteins. tion can occur between a peptidyl-tRNA located at the ribosomal Eukaryotic proteomes, such as that of Homo sapiens contain >7,000 P-site and an aminoacyl-tRNA in the A-site. However, the ribo- PPP-containing proteins and >15,000 XPPX-containing proteins some cannot form peptide bonds between all amino acids with that are all potential substrates for eIF-5A. thesameefficiency; this is exemplified by the amino acid proline, Results which has an imino group instead of a primary amino group in Δefp ΔyjeK ΔyjeA ΔyfcM other amino acids. Proline has been shown to be a particularly poor Proteomic Analysis of , , , and Strains. SILAC Δ Δ substrate for peptide-bond formation, both as a donor in the P-site was performed by growing the E. coli K-12 mutant ( efp, yjeK, Δ Δ Δ Δ andasanacceptorintheA-site(1–4). In fact, ribosomes stall when yjeA,or yfcM and lysA/ argA) strains in minimal medium “ ” –12 14 attempting to incorporate three or more consecutive proline resi- containing the light forms of lysine (K0 C6 N2) and arginine –12 14 dues (PPP) into the polypeptide chain (5–7). In this case, ribosome (R0 C6 N4), and the wild-type E. coli parental strain (MG1655 Δ Δ “ ” –13 15 stalling results from the slow rate of peptide-bond formation be- lysA argA)inthe heavy forms of lysine (K8 C6 N2)and –13 15 tween the peptidyl-Pro-Pro-tRNA located in the P-site and the arginine (R10 C6 N4). Cells were harvested at exponential Pro-tRNA in the A-site (6). In bacteria, the translational arrest growth phase, and the heavy-labeled (H) wild-type sample was is relieved by the translation elongation factor P (EF-P), which binds to the stalled ribosomes and stimulates peptide bond for- Significance mation (5, 6). In Escherichia coli, EF-P is posttranslationally modified by YjeA, YjeK, and YfcM (EpmA, EpmB, and EpmC) During protein synthesis, ribosomes catalyze peptide-bond (8–10) and the resulting lysinylation modification has been shown formation between amino acids with differing efficiency. We to be critical for the rescue activity of EF-P in vivo and in vitro show that two or more consecutive prolines induce ribosome (5, 6). EF-P homologs exist in all archaea and eukaryotes, termed stalling, and that stalling strength is influenced by the amino aIF-5A and eIF-5A, respectively (11). Yeast eIF-5A has recently acid preceding and following the prolines. In bacteria, the been shown to also rescue translational stalling at polyproline- elongation factor EF-P efficiently rescues the ribosome stalling stretches (12). Like EF-P, a/eIF-5A is also posttranslationally irrespective of the XPP or PPX motif. modified, but via hypusinylation rather than lysinylation (11). In addition to PPP, PPG triplets also induce ribosome stalling in Author contributions: K.J., J.R., and D.N.W. designed research; L.P., A.L.S., J.L., G.C.A., K.V., and M.S. performed research; L.P., A.L.S., J.L., G.C.A., K.V., M.S., T.T., K.J., J.R., and bacteria, which is rescued by EF-P (6). Moreover, a recent D.N.W. analyzed data; and D.N.W. wrote the paper. fi proteomics study identi ed APP, YIRYIR, and GSCGPG motifs The authors declare no conflict of interest. as conferring EF-P dependent translation (13). Ribosome stall- This article is a PNAS Direct Submission. ing has also been observed at PPA, PPD, PPE, PPN, PPW, APP, 1 To whom correspondence may be addressed. E-mail: [email protected] or and WPP triplets during in vitro translation (7), however it was not [email protected]. shown whether EF-P can relieve the translational arrest at these This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. triplets. Ribosome profiling data indicate ribosome accumulation 1073/pnas.1310642110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1310642110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 mixed with each light-labeled (L) mutant sample in 1:1 ratio of proteins related to translation and ATP synthesis, and up- based on their total protein concentration. The relative changes regulation of proteins related to chemotaxis, motility, and amino in protein levels were then determined by calculating the inver- acid biogenesis (Fig. S3). ted heavy/light (H/L) ratios for each mutant strain using MaxQuant (16). Our experimental setup resulted in 96 LC-MS measure- Down-Regulation of PPP-Containing Proteins in Δefp, ΔyjeK, and ments and 1,559,931 acquired MS/MS spectra. The database ΔyjeA Strains. Because EF-P was shown to enhance translation search led to identification of between 9,131 and 12,875 unique of polyproline-containing proteins (5, 6), we addressed whether peptide sequences in individual replicate experiments, with an down-regulation of polyproline-containing proteins due to the average absolute precursor ion mass accuracy of 0.33 ppm and absence of active EF-P was evident in the Δefp/ΔyjeK/ΔyjeA SD of 0.45 ppm. Identified peptides mapped to 2,098 protein datasets. In total, we detected 44 of the 96 possible polyproline- groups at 1% false discovery rate, comprising more than 48% of containing proteins in our dataset (Dataset S1), 28 of which were gene products in our sequence database (E. coli K-12 MG155). quantified in at least one replicate experiment and 21 of which This result is well in line with a recent work where a total of were quantified in both replicate experiments (Fig. 1E). As 2,118 proteins were detected, of which 1,984 were quantified in expected, we observed a marked shift in the distribution of protein E. coli grown in minimal medium (17). Of the 2,098 proteins ratios for the PPP-containing proteins in the Δefp/ΔyjeK/ΔyjeA, identified across all experiments, between 1,418 and 1,687 but not in the ΔyfcM data (Fig. 1 A–D): The median values for the were quantified (i.e., had two or more ratio counts in Max- PPP-containing protein normalized ratios were between −0.29 Quant analysis) in a single experiment, and these were therefore and −0.71 for Δefp/ΔyjeK/ΔyjeA compared with −0.04 and 0.04 for used for all subsequent analysis (Dataset S1).
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