Substrate-triggered position switching of TatA and TatB during Tat transport in <i>Escherichia coli</i>

The twin-arginine protein transport (Tat) machinery mediates the translocation of folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. The Escherichia coli Tat system comprises TatC and two additional sequence-related proteins, TatA and TatB. The active translocase is assembled on demand, with substrate-binding at a TatABC receptor complex triggering recruitment and assembly of multiple additional copies of TatA; however, the molecular interactions mediating translocase assembly are poorly understood. A 'polar cluster' site on TatC transmembrane (TM) helix 5 was previously identified as binding to TatB. Here, we use disulfide cross-linking and molecular modelling to identify a new binding site on TatC TM helix 6, adjacent to the polar cluster site. We demonstrate that TatA and TatB each have the capacity to bind at both TatC sites, however in vivo this is regulated according to the activation state of the complex. In the resting-state system, TatB binds the polar cluster site, with TatA occupying the TM helix 6 site. However when the system is activated by overproduction of a substrate, TatA and TatB switch binding sites. We propose that this substrate-triggered positional exchange is a key step in the assembly of an active Tat translocase

Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of <i>Mycobacterium tuberculosis</i>

Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic activity and for protein stability to thermal and chemical denaturation. We identified the intersubunit salt bridge Arg(80)-Asp(93) as essential for hexamer stability, compensating for the decreased intersubunit contact area. Breaking the salt bridge by the mutation D93N dramatically decreased protein thermal stability. The mutation also decreased stability to denaturation by urea and guanidinium. The D93N mutant was still hexameric and retained full activity. When exposed to low concentrations of urea it dissociated into folded monomers followed by unfolding while dissociation and unfolding of the wild type simultaneously occur at higher urea concentrations. The dissociation step was not observed in guanidine hydrochloride, suggesting that low concentration of salt may stabilize the hexamer. Indeed, guanidinium and many other salts stabilized the hexamer with a half maximum effect of about 0.1 M, increasing protein thermostability. The crystal structure of the D93N mutant has been solved

The TatC component of the twin-arginine protein translocase functions as an obligate oligomer

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild-type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild-type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.</p

In vitro translation of virally-encoded replication polyproteins to recapitulate polyprotein maturation processes

International audienceSingle-stranded, positive-sense RNA viruses encode essential replication polyproteins which are composed of several domains. They are usually subjected to finely regulated proteolytic maturation processes to generate cleavage intermediates and end-products. Both polyproteins and maturation products play multiple key roles that ultimately allow synthesis of viral genome progeny. Despite the importance of these proteins in the course of viral replication, their structural properties, including the conformational changes regulating their numerous functions, are poorly described at the structural level. This lack of information is mainly due to the extreme difficulty to express large, membrane-bound, multi-domain proteins with criteria suitable for structural biology methods. To tackle this challenge, we have used a wheat-germ cell-free expression system. We firstly establish that this approach allows to synthesize viral polyproteins encoded by two unrelated positive-sense RNA viruses, a human norovirus and a plant tymovirus. Then, we demonstrate that these polyproteins are fully functional and are spontaneously auto-cleaved by their active protease domain, giving rise to natural maturation products. Moreover, we show that introduction of point mutations in polyproteins allows to inhibit the proteolytic ma-turation process of each virus. This allowed us to express and partially purify the uncleaved full-length norovirus polyprotein and the tymoviral RNA-dependent RNA polymerase. Thus, this study provides a powerful tool to obtain soluble viral polyproteins and their maturation products in order to conduct challenging structural biology projects and therefore solve unanswered questions

Supplementary Figures and Tables from Substrate-triggered position switching of TatA and TatB during Tat transport in <i>Escherichia coli</i>

These are uploaded as a document along with the manuscript file

ATP synthase destabilization affects mitochondrial ultrastructure.

<p>72 hours after transduction with <i>Scramble</i> (A,B) or <i>ShATP5I</i> (C,D,E,F) lentiviral particles, adherent cells were fixed and observed by electron microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075429#s2" target="_blank">Materials and methods</a> (bar = 0.5 µm).</p

Steady state analysis of ATP synthase assembly in the absence of subunits <i>e</i> and <i>g</i>.

<p>(A) Mitochondrial proteins (50 µg) were separated by Tris-tricine SDS-PAGE. Subunits from the F₁ and the F₀ sectors were revealed by western blot using the appropriate antibodies (Sc: <i>Scramble</i>, Sh: <i>shATP5I</i>). (B) For each subunit, the signal was normalized to the signal of porin. Results obtained with <i>shATP5I</i> transduced cells were compared to control (<i>Scramble</i>) and are presented as a percentage. Measures are the mean of three different mitochondrial preparations (error bar: standard deviation).</p

Functional consequences of the depletion of subunits <i>e</i> and <i>g</i>.

<p>(A) Extracellular lactate concentration (µM) released by <i>Scramble</i> (white dot) and <i>ShATP5I</i> (black dot) was measured during cell growth (representative of 3 independent experiments). (B) Oxygen consumption flux were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075429#s2" target="_blank">Materials and methods</a> on adherent cells. O₂ flux consumption were measured in the presence of malate (0.5 M), pyruvate (0.5 M) and succinate (0.5 M) supplemented with oligomycin (0.5 µg⋅mL⁻¹) or CCCP (0.6 µM). Each measurement was performed three times in 5 minutes. Data are the mean of 4 experiments and are expressed as picomole of O₂ per minute per 10⁶ cells (black bar: <i>Scramble</i>; gray bar: <i>ShATP5I</i>; error bar: standard deviation; *: P<0.002). (C) Mitochondrial proteins (50 µg) were separated by Tris-glycine SDS-PAGE. Subunits representative for each respiratory complex were revealed by western blot using the appropriate antibodies (<i>Sc</i>: <i>Scramble</i>; <i>Sh; ShATP5I</i>). This figure is representative of three independent experiments.</p

ATP synthase destabilization induces fragmentation of the mitochondrial network.

<p>HeLa cells were transduced with either the <i>Scramble</i> or <i>ShATP5I</i> lentiviral particles. 72 hours after transduction, mitochondrial network morphology was observed on adherent cells by fluorescence microscopy. (A) Representative morphologies of the mitochondrial network classes (bar = 10 µm). (B) Percentages of the different classes in the two cellular populations. Cells were manually classified by two different persons on 200 randomly selected cells. Counts are the mean of 4 independent experiments (black bar: <i>Scramble</i>; gray bar: <i>ShATP5I</i>; error bar: standard deviation).</p

Human F₁F₀ ATP Synthase, Mitochondrial Ultrastructure and OXPHOS Impairment: A (Super-)Complex Matter?

<div><p>Mitochondrial morphogenesis is a key process of cell physiology. It is essential for the proper function of this double membrane-delimited organelle, as it ensures the packing of the inner membrane in a very ordered pattern called <i>cristae</i>. In yeast, the mitochondrial ATP synthase is able to form dimers that can assemble into oligomers. Two subunits (<i>e</i> and <i>g</i>) are involved in this supramolecular organization. Deletion of the genes encoding these subunits has no effect on the ATP synthase monomer assembly or activity and only affects its dimerization and oligomerization. Concomitantly, the absence of subunits <i>e</i> and <i>g</i> and thus, of ATP synthase supercomplexes, promotes the modification of mitochondrial ultrastructure suggesting that ATP synthase oligomerization is involved in <i>cristae</i> morphogenesis. We report here that in mammalian cells in culture, the shRNA-mediated down-regulation of subunits <i>e</i> and <i>g</i> affects the stability of ATP synthase and results in a 50% decrease of the available functional enzyme. Comparable to what was shown in yeast, when subunits <i>e</i> and <i>g</i> expression are repressed, ATP synthase dimers and oligomers are less abundant when assayed by native electrophoresis. Unexpectedly, mammalian ATP synthase dimerization/oligomerization impairment has functional consequences on the respiratory chain leading to a decrease in OXPHOS activity. Finally these structural and functional alterations of the ATP synthase have a strong impact on the organelle itself leading to the fission of the mitochondrial network and the disorganization of mitochondrial ultrastructure. Unlike what was shown in yeast, the impairment of the ATP synthase oligomerization process drastically affects mitochondrial ATP production. Thus we propose that mutations or deletions of genes encoding subunits <i>e</i> and <i>g</i> may have physiopathological implications.</p></div