Lauri Peil

Comparison and applications of label-free absolute proteome quantification methods on Escherichia coli.

Three different label-free proteome quantification methods--APEX, emPAI and iBAQ--were evaluated to measure proteome-wide protein concentrations in the cell. All the methods were applied to a sample from Escherichia coli chemostat culture. A Pearson squared correlation of approximately 0.6 among the three quantification methods was demonstrated. Importantly, the sum of quantified proteins by iBAQ and emPAI corresponded with the Lowry total protein quantification, demonstrating applicability of label-free methods for an accurate calculation of protein concentrations at the proteome level. The iBAQ method showed the best correlation between biological replicates, a normal distribution among all protein abundances, and the lowest variation among ribosomal protein abundances, which are expected to have equal amounts. Absolute quantitative proteome data enabled us to evaluate metabolic cost for protein synthesis and apparent catalytic activities of enzymes by integration with flux analysis. All the methods demonstrated similar ATP costs for protein synthesis for different cellular processes and that costs for expressing biomass synthesis related proteins were higher than those for energy generation. Importantly, catalytic activities of energy metabolism enzymes were an order or two higher than those of monomer synthesis. Interestingly, a staircase-like protein expression was demonstrated for most of the transcription units.

Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P

Significance During protein synthesis, ribosomes catalyze peptide-bond formation between amino acids with differing efficiency. We show that two or more consecutive prolines induce ribosome stalling, and that stalling strength is influenced by the amino acid preceding and following the prolines. In bacteria, the elongation factor EF-P efficiently rescues the ribosome stalling irrespective of the XPP or PPX motif. Ribosomes are the protein synthesizing factories of the cell, polymerizing polypeptide chains from their constituent amino acids. However, distinct combinations of amino acids, such as polyproline stretches, cannot be efficiently polymerized by ribosomes, leading to translational stalling. The stalled ribosomes are rescued by the translational elongation factor P (EF-P), which by stimulating peptide-bond formation allows translation to resume. Using metabolic stable isotope labeling and mass spectrometry, we demonstrate in vivo that EF-P is important for expression of not only polyproline-containing proteins, but also for specific subsets of proteins containing diprolyl motifs (XPP/PPX). Together with a systematic in vitro and in vivo analysis, we provide a distinct hierarchy of stalling triplets, ranging from strong stallers, such as PPP, DPP, and PPN to weak stallers, such as CPP, PPR, and PPH, all of which are substrates for EF-P. These findings provide mechanistic insight into how the characteristics of the specific amino acid substrates influence the fundamentals of peptide bond formation.

Lys34 of translation elongation factor EF-P is hydroxylated by YfcM

Lys34 of the conserved translation elongation factor P (EF-P) is post-translationally lysinylated by YjeK and YjeA--a modification that is critical for bacterial virulence. Here we show that the currently accepted Escherichia coli EF-P modification pathway is incomplete and lacks a final hydroxylation step mediated by YfcM, an enzyme distinct from deoxyhypusine hydroxylase that catalyzes the final maturation step of eukaryotic initiation factor 5A, the eukaryotic EF-P homolog.

Erythromycin- and Chloramphenicol-Induced Ribosomal Assembly Defects Are Secondary Effects of Protein Synthesis Inhibition

ABSTRACT Several protein synthesis inhibitors are known to inhibit ribosome assembly. This may be a consequence of direct binding of the antibiotic to ribosome precursor particles, or it could result indirectly from loss of coordination in the production of ribosomal components due to the inhibition of protein synthesis. Here we demonstrate that erythromycin and chloramphenicol, inhibitors of the large ribosomal subunit, affect the assembly of both the large and small subunits. Expression of a small erythromycin resistance peptide acting in cis on mature ribosomes relieves the erythromycin-mediated assembly defect for both subunits. Erythromycin treatment of bacteria expressing a mixture of erythromycin-sensitive and -resistant ribosomes produced comparable effects on subunit assembly. These results argue in favor of the view that erythromycin and chloramphenicol affect the assembly of the large ribosomal subunit indirectly.

Ribosome assembly in Escherichia coli strains lacking the RNA helicase DeaD/CsdA or DbpA

Ribosome subunit assembly in bacteria is a fast and efficient process. Among the nonribosomal proteins involved in ribosome biogenesis are RNA helicases. We describe ribosome biogenesis in Escherichia coli strains lacking RNA helicase DeaD (CsdA) or DbpA. Ribosome large subunit assembly intermediate particles (40S) accumulate at 25 °C and at 37 °C in the absence of DeaD but not without DbpA. 23S rRNA is incompletely processed in the 40S and 50S particles of the DeaD− strain. Pulse labeling showed that the 40S particles are converted nearly completely into functional ribosomes. The rate of large ribosomal subunit assembly was reduced about four times in DeaD‐deficient cells. Functional activity tests of the ribosomal particles demonstrated that the final step of 50S assembly, the activation step, was affected when DeaD was not present. The results are compatible with the model that predicts multiple DeaD‐catalyzed structural transitions of the ribosome large subunit assembly.

Identification of pseudouridine methyltransferase in Escherichia coli

In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem-loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m(3)Psi) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Psi1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m(3)Psi1915 is the only methylated pseudouridine in bacteria described to date.

Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site

The polymerization of amino acids into proteins occurs on ribosomes, with the rate influenced by the amino acids being polymerized. The imino acid proline is a poor donor and acceptor for peptide-bond formation, such that translational stalling occurs when three or more consecutive prolines (PPP) are encountered by the ribosome. In bacteria, stalling at PPP motifs is rescued by the elongation factor P (EF-P). Using SILAC mass spectrometry of Escherichia coli strains, we identified a subset of PPP-containing proteins for which the expression patterns remained unchanged or even appeared up-regulated in the absence of EF-P. Subsequent analysis using in vitro and in vivo reporter assays revealed that stalling at PPP motifs is influenced by the sequence context upstream of the stall site. Specifically, the presence of amino acids such as Cys and Thr preceding the stall site suppressed stalling at PPP motifs, whereas amino acids like Arg and His promoted stalling. In addition to providing fundamental insight into the mechanism of peptide-bond formation, our findings suggest how the sequence context of polyproline-containing proteins can be modulated to maximize the efficiency and yield of protein production.

A proteomics approach reveals divergent molecular responses to salinity in populations of European whitefish (Coregonus lavaretus)

Osmoregulation is a vital physiological function for fish, as it helps maintain a stable intracellular concentration of ions in environments of variable salinities. We focused on a primarily freshwater species, the European whitefish (Coregonus lavaretus), to investigate the molecular mechanisms underlying salinity tolerance and examine whether these mechanisms differ between genetically similar populations that spawn in freshwater vs. brackishwater environments. A common garden experiment involving 27 families in two populations and five salinity treatments together with a large‐scale, high‐resolution mass spectrometry experiment that quantified 1500 proteins was conducted to assess phenotypic and proteomic responses during early development, from fertilization until hatching, in the studied populations. The populations displayed drastically different phenotypic and proteomic responses to salinity. Freshwater‐spawning whitefish showed a significantly higher mortality rate in higher salinity treatments. Calcium, an ion involved in osmotic stress sensing, had a central role in the observed proteomic responses. Brackishwater‐spawning fish were capable of viable osmoregulation, which was modulated by cortisol, an important seawater‐adaptation hormone in teleost fish. Several proteins were identified to play key roles in osmoregulation, most importantly a highly conserved cytokine, tumour necrosis factor, whereas calcium receptor activities were associated with salinity adaptation. These results imply that individuals from these populations are most likely adapted to their local environments, even though the baseline level of genetic divergence between them is low (FST = 0.049). They also provide clues for choosing candidate loci for studying the molecular basis of salinity adaptation in other species. Further, our approach provides an example of how proteomic methods can be successfully used to obtain novel insights into the molecular mechanisms behind adaptation in non‐model organism.

Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits

During the last step in 40S ribosome subunit biogenesis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously characterized pre-40S components, but retain the endonuclease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in actively growing cells. Nob1 and Pno1 bind overlapping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Binding sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation.

Spectral counting label-free proteomics.

Label-free proteome quantification methods used in bottom-up mass-spectrometry based proteomics are gaining more popularity as they are easy to apply and can be integrated into different workflows without any extra effort or cost. In the label-free proteome quantification approach, samples of interest are prepared and analyzed separately. Mass-spectrometry is generally not recognized as a quantitative method as the ionization efficiency of peptides is dependent on composition of peptides. Label-free quantification methods have to overcome this limitation by additional computational calculations. There are several algorithms available that take into account the sequence and length of the peptides and compute the predicted abundance of proteins in the sample. Label-free methods can be divided into two categories: peptide peak intensity based quantification and spectral counting quantification that relies on the number of peptides identified from a given protein.This protocol will concentrate on spectral counting quantification-exponentially modified protein abundance index (emPAI). Normalized emPAI, most commonly derived from Mascot search results, can be used for broad comparison of entire proteomes. Absolute quantification of proteins based on emPAI values with or without added standards will be demonstrated. Guidelines will be given on how to easily integrate emPAI into existing data; for example, calculating emPAI based absolute protein abundances from iTRAQ data without added standards.