Membrane Protein Biogenesis in Ffh- or FtsY-Depleted Escherichia coli

BACKGROUND: The Escherichia coli version of the mammalian signal recognition particle (SRP) system is required for biogenesis of membrane proteins and contains two essential proteins: the SRP subunit Ffh and the SRP-receptor FtsY. Scattered in vivo studies have raised the possibility that expression of membrane proteins is inhibited in cells depleted of FtsY, whereas Ffh-depletion only affects their assembly. These differential results are surprising in light of the proposed model that FtsY and Ffh play a role in the same pathway of ribosome targeting to the membrane. Therefore, we decided to evaluate these unexpected results systematically. METHODOLOGY/PRINCIPAL FINDINGS: We characterized the following aspects of membrane protein biogenesis under conditions of either FtsY- or Ffh-depletion: (i) Protein expression, stability and localization; (ii) mRNA levels; (iii) folding and activity. With FtsY, we show that it is specifically required for expression of membrane proteins. Since no changes in mRNA levels or membrane protein stability were detected in cells depleted of FtsY, we propose that its depletion may lead to specific inhibition of translation of membrane proteins. Surprisingly, although FtsY and Ffh function in the same pathway, depletion of Ffh did not affect membrane protein expression or localization. CONCLUSIONS: Our results suggest that indeed, while FtsY-depletion affects earlier steps in the pathway (possibly translation), Ffh-depletion disrupts membrane protein biogenesis later during the targeting pathway by preventing their functional assembly in the membrane

Phenological shifts of abiotic events, producers and consumers across a continent

Ongoing climate change can shift organism phenology in ways that vary depending on species, habitats and climate factors studied. To probe for large-scale patterns in associated phenological change, we use 70,709 observations from six decades of systematic monitoring across the former Union of Soviet Socialist Republics. Among 110 phenological events related to plants, birds, insects, amphibians and fungi, we find a mosaic of change, defying simple predictions of earlier springs, later autumns and stronger changes at higher latitudes and elevations. Site mean temperature emerged as a strong predictor of local phenology, but the magnitude and direction of change varied with trophic level and the relative timing of an event. Beyond temperature-associated variation, we uncover high variation among both sites and years, with some sites being characterized by disproportionately long seasons and others by short ones. Our findings emphasize concerns regarding ecosystem integrity and highlight the difficulty of predicting climate change outcomes. The authors use systematic monitoring across the former USSR to investigate phenological changes across taxa. The long-term mean temperature of a site emerged as a strong predictor of phenological change, with further imprints of trophic level, event timing, site, year and biotic interactions.Peer reviewe

Evidence for a cytoplasmic pool of ribosome-free mRNAs encoding inner membrane proteins in Escherichia coli.

Translation-independent mRNA localization represents an emerging concept in cell biology. In Escherichia coli, mRNAs encoding integral membrane proteins (MPRs) are targeted to the membrane where they are translated by membrane associated ribosomes and the produced proteins are inserted into the membrane co-translationally. In order to better understand aspects of the biogenesis and localization of MPRs, we investigated their subcellular distribution using cell fractionation, RNA-seq and qPCR. The results show that MPRs are overrepresented in the membrane fraction, as expected, and depletion of the signal recognition particle-receptor, FtsY reduced the amounts of all mRNAs on the membrane. Surprisingly, however, MPRs were also found relatively abundant in the soluble ribosome-free fraction and their amount in this fraction is increased upon overexpression of CspE, which was recently shown to interact with MPRs. CspE also conferred a positive effect on the membrane-expression of integral membrane proteins. We discuss the possibility that the effects of CspE overexpression may link the intriguing subcellular localization of MPRs to the cytosolic ribosome-free fraction with their translation into membrane proteins and that the ribosome-free pool of MPRs may represent a stage during their targeting to the membrane, which precedes translation

Characterization of 4 model untranslatable RNAs.

<p><b>(A)</b> Schematic representation of the Ra-Rd encoding genes [see text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134413#pone.0134413.s001" target="_blank">S1 Fig</a>]. <b>(B)</b> Uracil content of the model transcripts, utilizing a sliding window of 55 nucleotides as calculated by the software DNA Strider. <b>(C)</b> Wild type <i>E</i>. <i>coli</i> expressing Ra or Rb were disrupted by sonication and cell extracts were fractionated by sucrose density gradient (a representative gradient is shown). The gradient fractions were analyzed for RNA content (A₂₆₀). The indicated free and ribosomal fractions were pooled. <b>(D)</b> The contents of the indicated R transcripts and endogenous, translatable transcripts encoding PrfA and RpoD as controls, were measured by qPCR in the pooled free and ribosomal fractions. Error bars indicate SEM (n = 3).</p

Co-sedimentation of Ffh with 70S ribosome from cells depleted of FtsY.

<p><i>E. coli</i> FJP10 harboring pT7-5/LacY were grown for 6.5 h with or without 0.05% arabinose (+/−ara). (A) Cells were fractionated and samples of extracts (E, 10 µg), supernatants (S, 10 µg) and pellets after ultracentrifugation (P, 4.5 µg) were analyzed by Western blotting. The Ffh band was quantified by densitometry. (B, C) The ultracentrifugation pellets (containing cytosolic ribosomes and membranes) were separated by sucrose gradient (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009130#s4" target="_blank">Material and Methods</a>). (B) Optical density at 260 nm of each fraction. (C) Western blotting of each fraction with antibodies against the ribosomal protein L9, LacY, and Ffh.</p

Characterization of 4 model untranslatable RNAs.

<p><b>(A)</b> Schematic representation of the Ra-Rd encoding genes [see text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134413#pone.0134413.s001" target="_blank">S1 Fig</a>]. <b>(B)</b> Uracil content of the model transcripts, utilizing a sliding window of 55 nucleotides as calculated by the software DNA Strider. <b>(C)</b> Wild type <i>E</i>. <i>coli</i> expressing Ra or Rb were disrupted by sonication and cell extracts were fractionated by sucrose density gradient (a representative gradient is shown). The gradient fractions were analyzed for RNA content (A₂₆₀). The indicated free and ribosomal fractions were pooled. <b>(D)</b> The contents of the indicated R transcripts and endogenous, translatable transcripts encoding PrfA and RpoD as controls, were measured by qPCR in the pooled free and ribosomal fractions. Error bars indicate SEM (n = 3).</p

Effect of Ffh-depletion on the localization and functional assembly of MdfA.

<p>(A) Extracts from Ffh-depleted or non-depleted WAM121 cells harboring pT7-5/Mdf379-PhoA were fractionated (E, extracts; S, supernatant; P, pellets). Examination of MdfA379-PhoA in the different fractions was accomplished by Western blotting. (B) Membranes isolated by floatation were collected by ultracentrifugation without and with pre-treatment with 0.2 M Na₂CO₃ (pH 11) and analyzed by Western blotting using anti-PhoA and Anti-SecA antibodies. (C) The alkaline phosphatase activity of MdfA379-PhoA in WAM121 cells is presented as the rate of p-nitrophenyl phosphate hydrolysis. The experiments were repeated 3 times and error bars indicate standard deviations. (C) Efflux activity of MdfA in Ffh-depleted or non-depleted cells was measured by following EtBr fluorescence. The experiments were repeated at least 3 times, and the results shown are representative.</p