Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

Photosystem II photoinactivation, repair, and protection in marine centric diatoms.

Diatoms are important contributors to aquatic primary production, and can dominate phytoplankton communities under variable light regimes. We grew two marine diatoms, the small Thalassiosira pseudonana and the large Coscinodiscus radiatus, across a range of temperatures and treated them with a light challenge to understand their exploitation of variable light environments. In the smaller T. pseudonana, photosystem II (PSII) photoinactivation outran the clearance of PSII protein subunits, particularly in cells grown at sub- or supraoptimal temperatures. In turn the absorption cross section serving PSII photochemistry was down-regulated in T. pseudonana through induction of a sustained phase of nonphotochemical quenching that relaxed only slowly over 30 min of subsequent low-light incubation. In contrast, in the larger diatom C. radiatus, PSII subunit turnover was sufficient to counteract a lower intrinsic susceptibility to photoinactivation, and C. radiatus thus did not need to induce sustained nonphotochemical quenching under the high-light treatment. T. pseudonana thus incurs an opportunity cost of sustained photosynthetic down-regulation after the end of an upward light shift, whereas the larger C. radiatus can maintain a balanced PSII repair cycle under comparable conditions.

The harmful alga Aureococcus anophagefferens utilizes 19'-butanoyloxyfucoxanthin as well as xanthophyll cycle carotenoids in acclimating to higher light intensities.

Aureococcus anophagefferens is a picoplanktonic microalga that is very well adapted to growth at low nutrient and low light levels, causing devastating blooms ("brown tides") in estuarine waters. To study the factors involved in long-term acclimation to different light intensities, cells were acclimated for a number of generations to growth under low light (20µmolphotonsm(-2)s(-1)), medium light (60 or 90µmolphotonsm(-2)s(-1)) and high light (200µmolphotonsm(-2)s(-1)), and were analyzed for their contents of xanthophyll cycle carotenoids (the D pool), fucoxanthin and its derivatives (the F pool), Chls c(2) and c(3), and fucoxanthin Chl a/c polypeptides (FCPs). Higher growth light intensities resulted in increased steady state levels of both diadinoxanthin and diatoxanthin. However, it also resulted in the conversion of a significant fraction of fucoxanthin to 19'-butanoyloxyfucoxanthin without a change in the total F pool. The increase in 19'-butanoyloxyfucoxanthin was paralleled by a decrease in the effective antenna size, determined from the slope of the change in F(0) as a function of increasing light intensity. Transfer of acclimated cultures to a higher light intensity showed that the conversion of fucoxanthin to its derivative was a relatively slow process (time-frame of hours). We suggest the replacement of fucoxanthin with the bulkier 19'-butanoyloxyfucoxanthin results in a decrease in the light-harvesting efficiency of the FCP antenna and is part of the long-term acclimative response to growth at higher light intensities.

High light stress and the one-helix LHC-like proteins of the cryptophyte Guillardia theta.

Cryptophytes like the cryptomonad Guillardia theta are part of the marine phytoplankton and therefore major players in global carbon and biogeochemical cycles. Despite the importance for the cell in being able to cope with large changes in illumination on a daily basis, very little is known about photoprotection mechanisms in cryptophytes. Here, we show that Guillardia theta is able to perform non-photochemical quenching, although none of the usual xanthophyll cycle pigments (e.g., zeaxanthin, diadinoxanthin, diatoxanthin) are present at detectable levels. Instead, acclimation to high light intensity seems to involve an increase of alloxanthin. Guillardia theta has genes for 2 one-helix "light-harvesting-like" proteins, related to some cyanobacterial genes which are induced in response to high light stress. Both the plastid-encoded gene (hlipP) and the nucleomorph-encoded gene (HlipNm) are expressed, but transcript levels decrease rather than increase during high light exposure, suggesting that they are not involved in a high light stress response. The HlipNm protein was detected with a specific antibody; expression was constant, independent of the light exposure.

Structure, binding, and activity of Syd, a SecY-interacting protein.

The Syd protein has been implicated in the Sec-dependent transport of polypeptides across the bacterial inner membrane. Using Nanodiscs, we here provide direct evidence that Syd binds the SecY complex, and we demonstrate that interaction involves the two electropositive and cytosolic loops of the SecY subunit. We solve the crystal structure of Syd and together with cysteine cross-link analysis, we show that a conserved concave and electronegative groove constitutes the SecY-binding site. At the membrane, Syd decreases the activity of the translocon containing loosely associated SecY-SecE subunits, whereas in detergent solution Syd disrupts the SecYEG heterotrimeric associations. These results support the role of Syd in proofreading the SecY complex biogenesis and point to the electrostatic nature of the Sec channel interaction with its cytosolic partners.

New analysis of the toxic compounds from the Androctonus mauretanicus mauretanicus scorpion venom.

Scorpion venoms are very complex mixtures of molecules, most of which are peptides displaying different kinds of biological activity. Indeed, these peptides specifically bind to a variety of pharmacological targets, in particular ionic channels located in prey tissues, resulting in neurotoxic effects. Toxins modulating Na+, K+, Ca2+ and Cl(-) currents have been described in scorpion venoms. In this work, we have used several specific antibodies raised against the most lethal scorpion toxins already described to screen the Moroccan scorpion Androctonus mauretanicus mauretanicus venom in order to characterize new compounds. This immunological screening was also implemented by toxicity tests in mice and with mass spectrometry study, providing new informations on the molecular composition of this venom. In fine, we were able to determine the molecular masses of 70-80 different compounds. According to the immunological data obtained, many toxins cross-react with three sera raised against the most lethal alpha-toxins found in North African scorpion venoms, but not at all with those raised against the main beta-toxins from South and North American venoms. Some of the previously described toxins from Androctonus mauretanicus mauretanicus venom could thus be detected by combining immunological tests, toxicity in mice and molecular masses. Among these toxins, one of them, which showed a mild cross-reaction with the serum raised against AaH I (a highly potent toxin from the venom of Androctonus australis), was identified as Amm III and fully sequenced.

Cysteine scanning mutagenesis and topological mapping of the Escherichia coli twin-arginine translocase TatC Component.

The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.

Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA.

The translocon is a membrane-embedded protein assembly that catalyzes protein movement across membranes. The core translocon, the SecYEG complex, forms oligomers, but the protein-conducting channel is at the center of the monomer. Defining the properties of the SecYEG protomer is thus crucial to understand the underlying function of oligomerization. We report here the reconstitution of a single SecYEG complex into nano-scale lipid bilayers, termed Nanodiscs. These water-soluble particles allow one to probe the interactions of the SecYEG complex with its cytosolic partner, the SecA dimer, in a membrane-like environment. The results show that the SecYEG complex triggers dissociation of the SecA dimer, associates only with the SecA monomer and suffices to (pre)-activate the SecA ATPase. Acidic lipids surrounding the SecYEG complex also contribute to the binding affinity and activation of SecA, whereas mutations in the largest cytosolic loop of the SecY subunit, known to abolish the translocation reaction, disrupt both the binding and activation of SecA. Altogether, the results define the fundamental contribution of the SecYEG protomer in the translocation subreactions and illustrate the power of nanoscale lipid bilayers in analyzing the dynamics occurring at the membrane.

The entire N-terminal half of TatC is involved in twin-arginine precursor binding.

Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding.

Genomic characterisation of the toxin Amm VIII from the scorpion Androctonus mauretanicus mauretanicus.

The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166 bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.

Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli.

The twin-arginine translocation (Tat) machinery of the Escherichia coli inner membrane is dedicated to the export of proteins harboring a conserved SRRxFLK motif in their signal sequence. TatA, TatB, and TatC are the functionally essential constituents of the Tat machinery, but their precise function is unknown. Using site-specific crosslinking, we have analyzed interactions of the twin-arginine precursor preSufI with the Tat proteins upon targeting to inner membrane vesicles. TatA association is observed only in the presence of a transmembrane H(+) gradient. TatB is found in contact with the entire signal sequence and adjacent parts of mature SufI. Interaction of TatC with preSufI is, however, restricted to a discrete area around the consensus motif. The results reveal a hierarchy in targeting of a Tat substrate such that for the primary interaction, TatC is both necessary and sufficient while a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore.

Characterization of Amm VIII from Androctonus mauretanicus mauretanicus: a new scorpion toxin that discriminates between neuronal and skeletal sodium channels.

The venom of the scorpion Androctonus mauretanicus mauretanicus was screened by use of a specific serum directed against AaH II, the scorpion alpha-toxin of reference, with the aim of identifying new analogues. This led to the isolation of Amm VIII (7382.57 Da), which gave a highly positive response in ELISA, but was totally devoid of toxicity when injected subcutaneously into mice. In voltage-clamp experiments with rat brain type II Na+ channel rNa(v)1.2 or rat skeletal muscle Na+ channel rNa(v)1.4, expressed in Xenopus oocytes, the EC50 values of the toxin-induced slowing of inactivation were: 29+/-5 and 416+/-14 nM respectively for AmmVIII and 2.6+/-0.3 nM and 2.2+/-0.2 nM, respectively, for AaH II interactions. Accordingly, Amm VIII clearly discriminates neuronal versus muscular Na+ channel. The Amm VIII cDNA was amplified from a venom gland cDNA library and its oligonucleotide sequence determined. It shows 87% sequence homology with AaH II, but carries an unusual extension at its C-terminal end, consisting of an additional Asp due to a point mutation in the cDNA penultimate codon. We hypothesized that this extra amino acid residue could induce steric hindrance and dramatically reduce recognition of the target by Amm VIII. We constructed a model of Amm VIII based on the X-ray structure of AaH II to clarify this point. Molecular modelling showed that this C-terminal extension does not lead to an overall conformational change in Amm VIII, but drastically modifies the charge repartition and, consequently, the electrostatic dipole moment of the molecule. At last, liquid-phase radioimmunassays with poly- and monoclonal anti-(AaH II) antibodies showed the loss of conformational epitopes between AaH II and Amm VIII.

Influence of tat mutations on the ribose-binding protein translocation in Escherichia coli.

Proteins are exported across the bacterial cytoplasmic membrane either as unfolded precursors via the Sec machinery or in folded conformation via the Tat system. The ribose-binding protein (RBP) of Escherichia coli is a Sec-pathway substrate. Intriguingly, it exhibits fast folding kinetics and its export is independent of SecB, a general chaperone protein dedicated for protein secretion. In this study, we found that the quantity of RBP was significantly reduced in the periplasm of tat mutants, which was restored by in trans expression of the tatABC genes. Pulse-chase experiments showed that significant amount of wild-type RBP was processed in a secY mutant in the presence of azide (SecA inhibitor), whereas the processing of a slow folding RBP derivative was almost completely blocked under the same conditions. These results would suggest that under the Sec-defective conditions the export of a portion of folded RBP could be rescued by the Tat system.

Expanding the scorpion toxin alpha-KTX 15 family with AmmTX3 from Androctonus mauretanicus.

A novel toxin, AmmTX3 (3823.5 Da), was isolated from the venom of the scorpion Androctonus mauretanicus. It showed 94% sequence homology with Aa1 from Androctonus australis and 91% with BmTX3 from Buthus martensi which, respectively, block A-type K+ current in cerebellum granular cells and striatum cultured neurons. Binding and displacement experiments using rat brain synaptosomes showed that AmmTX3 and Aa1 competed effectively with 125I-labelled sBmTX3 binding. They fully inhibited the 125I-labelled sBmTX3 binding (Ki values of 19.5 pm and 44.2 pm, respectively), demonstrating unambiguously that the three molecules shared the same target in rat brain. The specific binding parameters of 125I-labelled AmmTX3 for its site were determined at equilibrium (Kd = 66 pm, Bmax = 22 fmol per mg of protein). Finally, patch-clamp experiments on striatal neurons in culture demonstrated that AmmTX3 was able to inhibit the A-type K+ current (Ki = 131 nm).

Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes.

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.

Separate analysis of twin-arginine translocation (Tat)-specific membrane binding and translocation in Escherichia coli.

The twin-arginine translocation (Tat) pathway exports those precursor proteins to the periplasmic space of bacteria that harbor a twin-arginine (RR) consensus motif in their signal sequences. We have reproduced translocation of several Tat substrates into inside-out plasma membrane vesicles from Escherichia coli. Translocation proceeding at an efficiency of up to 20% occurs specifically via the Tat pathway as indicated by (i) its requirement for elevated levels of the TatABC proteins in the membrane vesicles, (ii) competition by an intact twin-arginine signal peptide, and (iii) susceptibility toward dissipation of the transmembrane H(+) gradient. The latter treatment, while blocking translocation, still allows for functional membrane association of Tat precursors. This is shown by the finding that translocation of isolated membrane-bound Tat precursor is restored upon re-energization of the vesicles.