Novel Transporters from Hemiascomycete Yeasts

The gene for subunit II of cytochrome oxidase in the yeast Hansenula saturnus was previously shown to be located on a 1.7 kb HindIII-BamHI fragment of mitochondrial DNA (Lawson and Deters, accompanying paper). In this paper, we report the nucleotide sequence of a large part of this fragment, covering the coding region of the subunit II gene, designated coxII, and its 5' and 3' flanking regions. The coding region of the coxII gene consists of a continuous open reading frame, 744 nucleotides long, containing 6 in frame TGA codons. Examination of the sequence and alignment with known homologous gene sequences of other organisms indicates that TGA codes for tryptophan in H. saturnus mitochondria as it does in several other mitochondria. Despite considerable homology to subunit II of Saccharomyces cerevisiae, there are 9 codons used in coxII that are not used in the corresponding S. cerevisiae gene. CTT, which is believed to code for threonine in S. cerevisiae mitochondria, appears 3 times in coxII and probably codes for leucine. While the CGN family is rarely, if ever, used in S. cerevisiae mitochondria, CGT appears 4 times in coxII and probably codes for arginine. The deduced amino acid sequence, excluding the first ten amino acids at the N-terminus, is 81% homologous to the amino acid sequence of the S. cerevisiae subunit II protein. The first ten amino acids at the N-terminus are not homologous to the N-terminus of the S. cerevisiae protein but are highly homologous to the first ten amino acids of the deduced amino acid sequence of subunit II of Neurospora crassa. Minor variations of a transcription initiation signal and an end of message or processing signal reported in S. cerevisiae are found in the regions flanking the H. saturnus coxII gene. The subunit II gene contains numerous symmetrical elements, i.e. palindromes, inverted repeats, and direct repeats. Some of these have conserved counterparts in the S. cerevisiae subunit II gene, suggesting that they may be functionally or structurally important.

Purification and identification of antioxidant and ACE-inhibitory peptide from Saccharomyces cerevisiae protein hydrolysate

Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The "activated" Ub, linked via a thioester bond to a specific cysteine residue in one of several Ub-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins. In the yeast Saccharomyces cerevisiae, a 114-kDa E1 enzyme is encoded by an essential gene termed UBA1 (McGrath, J.P., Jentsch, S., and Varshavsky, A. (1991) EMBO J. 10, 227-236). We describe the isolation and analysis of another essential gene, termed UBA2, that encodes a 71-kDa protein with extensive sequence similarities to both the UBA1-encoded yeast E1 and E1 enzymes of other organisms. The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserved between the known E1 enzymes and contains the active-site cysteine of E1. This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involved a transient thioester bond between Uba2p and either Ub or another protein. Uba2p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Uba1p (E1 enzyme) and Uba2p cannot complement each others essential functions even if their subcellular localization is altered by mutagenesis. Uba2p appears to interact with itself and several other S. cerevisiae proteins with apparent molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitinated in vivo, suggesting that at least a fraction of Uba2p is metabolically unstable. Uba2p is likely to be a component of the Ub system that functions as either an E2 or E1/E2 enzyme.

The Génolevures online database (http://cbi.labri.fr/Genolevures/) provides data and tools to facilitate comparative genomic studies on hemiascomycetous yeasts. Now, four complete genome sequences recently determined (Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica) have been added to the partial sequences of 13 species previously analysed by a random approach. The database also includes the reference genome Saccharomyces cerevisiae. Data are presented with a focus on relations between genes and genomes: conservation of genes and gene families, speciation, chromosomal reorganization and synteny. The Génolevures site includes a community area for specific studies by members of the international community.

SbmA is a membrane transporter from Escherichia coli that imports antimicrobial peptides. SbmA belongs to the SbmA-like peptide transporter (SLiPT) family. Although the protein is a secondary active transporter that is energized by the proton gradient, it is structurally related to the transmembrane domain (TMD) of ATP-binding cassette (ABC) transporters. SbmA therefore bridges the structural divide between primary and secondary transporters. However, it remains unclear, if SbmA also shares the mechanism of alternating access with ABC transporters, because only a single (outward-open) state is resolved. Here, we show by sequence analysis that SbmA is likely evolved from the TMD of an early ancestor of the ABC transporter YddA. We determine the cryogenic electron microscopy structures of SbmA in occluded and inward-facing states. These conformations closely resemble equivalent states found in ABC transporters, indicating a shared structural mechanism of transport. In contrast to ABC transporters, where nucleotide binding, hydrolysis and release steer conformational changes necessary for substrate translocation, electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations reveal how pH changes induce conformational transitions in SbmA, consistent with a mechanism of substrate internalization that utilizes the transmembrane proton gradient.

BackgroundWe recently reported that bacterial lipopolysaccharide (LPS)-induced inflammation decreases the expression of the primary thyroid hormone transporters at the blood–brain barrier, organic anion-transporting polypeptide 1c1 (OATP1c1) and monocarboxylate transporter 8 (MCT8). l-type amino acid transporters 1 and 2 (LAT1 & LAT2) are regarded as secondary thyroid hormone transporters, and are expressed in cells of the blood–brain or blood-cerebrospinal fluid barrier and by neurons. The purpose of this study was to examine the effect of LPS-induced inflammation on the expression of LAT1 and LAT2, as these may compensate for the downregulation of OATP1c1 and MCT8.MethodsLPS (2.5 mg/kg body weight) was injected intraperitoneally to adult, male, Sprague–Dawley rats and C57Bl/6 mice, which were euthanized 2, 4, 9, 24 or 48 h later. LAT1 and LAT2 mRNA expression were studied on forebrain sections using semiquantitative radioactive in situ hybridization. LAT1 protein levels in brain vessels were studied using LAT1 immunofluorescence. Statistical comparisons were made by the non-parametric Kruskal–Wallis and Dunn’s tests.ResultsIn both species, LAT1 mRNA decreased in brain blood vessels as soon as 2 h after LPS injection and was virtually undetectable at 4 h and 9 h. During recovery from endotoxemia, 48 h after LPS injection, LAT1 mRNA in brain vessels increased above control levels. A modest but significant decrease in LAT1 protein levels was detected in the brain vessels of mice at 24 h following LPS injection. LPS did not affect LAT1 and LAT2 mRNA expression in neurons and choroid plexus epithelial cells.ConclusionsThe results demonstrate that LPS-induced inflammation rapidly decreases LAT1 mRNA expression at the blood–brain barrier in a very similar manner to primary thyroid hormone transporters, while changes in LAT1 protein level follow a slower kinetics. The data raise the possibility that inflammation may similarly down-regulate other blood–brain barrier transport systems at the transcriptional level. Future studies are required to examine this possibility and the potential pathophysiological consequences of inflammation-induced changes in blood–brain barrier transport functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-015-0016-8) contains supplementary material, which is available to authorized users.

Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism.

Sequence similarity is probably the most widely used tool to infer functional linkage between proteins. The fully sequenced, much researched, genome of Saccharomyces cerevisiae gives us on opportunity to compare and statistically quantify computational methods based on sequence similarity, which aim to detect such linkage. In addition, the amount of data regarding Saccharomyces Cerevisiae genes and proteins, which is not directly based on sequence is rapidly increasing. Consequently, it allows investigation of the connections and correlation between classification based on these types of data and that based solely on sequence similarity. In this work we start with a simple clustering algorithm to cluster genes based on the BLAST E-score of their similarity. We analyze how well one can infer function from these clusters and for how many of the genes that are currently unknown one can suggest a prediction. Given these parameters, we show that even a simple algorithm achieves better results than simply considering the BLAST output of matching genes. In the second part of the paper, we show that there is a highly significant correlation (p-value < 10(-4) for the vast majority of the experiments) between the aforementioned clusters and other types of classifications. Namely, we show that a pair of genes being clustered together is correlated with these genes having similar expression patterns in DNA array experiments and with the encoded proteins being involved in protein-protein interactions. Although this correlation is highly significant, it is, of course, not strong enough to be, by itself, a tool for predicting co-regulation of genes or interaction of proteins. We discuss possible explanations for this correlation. Furthermore, the statistical evaluation of these results should be considered when developing tools that are aimed at making such predictions.

Mutations at the flightless-I locus (fliI) of Drosophila melanogaster cause flightlessness or, when severe, incomplete cellularization during early embryogenesis, with subsequent abnormalities in mesoderm invagination and in gastrulation. After chromosome walking, deficiency mapping, and transgenic analysis, we have isolated and characterized flightless-I cDNAs, enabling prediction of the complete amino acid sequence of the 1256-residue protein. Data base searches revealed a homologous gene in Caenorhabditis elegans, and we have isolated and characterized corresponding cDNAs. By using the polymerase chain reaction with nested sets of degenerate oligonucleotide primers based on conserved regions of the C. elegans and D. melanogaster proteins, we have cloned a homologous human cDNA. The predicted C. elegans and human proteins are, respectively, 49% and 58% identical to the D. melanogaster protein. The predicted proteins have significant sequence similarity to the actin-binding protein gelsolin and related proteins and, in addition, have an N-terminal domain consisting of a repetitive amphipathic leucine-rich motif. This repeat is found in D. melanogaster, Saccharomyces cerevisiae, and mammalian proteins known to be involved in cell adhesion and in binding to other proteins. The structure of the maternally expressed flightless-I protein suggests that it may play a key role in embryonic cellularization by interacting with both the cytoskeleton and other cellular components. The presence of a highly conserved homologue in nematodes, flies, and humans is indicative of a fundamental role for this protein in many metazoans.

Thorough analysis of the cta operon of Synechocystis sp. PCC6803 (grown in high-concentration salt medium to enhance the expression of respiratory proteins) showed that, apart from ctaCDE and Fb genes potentially encoding subunits I, II, III, and a small pseudo-bacteria-like subunit-IV of unknown function, a large mitochondria-like cta-Fm gene and a pronounced terminator structure are additional components of the operon. The deduced cta Fm gene product shows approximately 50% and 20% sequence identity to the Saccharomyces cerevisiae and beef heart mitochondrial COIV proteins, respectively. It also shows amino acid regions (near the N terminus, on the cytosolic side) with conspicuous sequence similarities to adenylate-binding proteins such as ATP synthase beta subunit Walker A and B consensus regions or to adenylate kinase. We suggest that, similar to the situation with beef heart mitochondria, it is the mitochondria-like subunit-IV of the cyanobacterial aa3-type cytochrome-c oxidase that confers allosteric properties to the cyanobacterial enzyme, the H+/e- ratios of cytochrome c oxidation being significantly lowered by ATP (intravesicular or intraliposomal) but enhanced by ADP. Therefore, the antagonistic action of ATP and ADP was in a way that the redox reaction proper, was always significantly less affected than the coupled proton translocation. Evolutionary and ecological implications of the unusual allosteric regulation of a prokaryotic cytochrome-c oxidase is discussed.

Watching the Pulleys Turn while the Elevator Moves in a Secondary Transporter

A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thalianahas recently been cloned, isolated, and characterized [Garcia-Ortiz, M. V., Ariza, R. R., and Roldan-Arjona, T. (2001) Plant Mol. Biol. 47, 795-804]. This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster, and human Ogg1 proteins. We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine-derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the 17 modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions. Excision depended on incubation time, enzyme concentration, and substrate concentration and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins. The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics.

Zinc can occur in extremely high concentrations in acidic, heavy metal polluted environments inhabited by acidophilic prokaryotes. Although these organisms are able to thrive in such severely contaminated ecosystems their resistance mechanisms have not been well studied. Bioinformatic analysis of a range of acidophilic bacterial and archaeal genomes identified homologues of several known zinc homeostasis systems. These included primary and secondary transporters, such as the primary heavy metal exporter ZntA and Nramp super-family secondary importer MntH. Three acidophilic model microorganisms, the archaeon 'Ferroplasma acidarmanus', the Gram negative bacterium Acidithiobacillus caldus, and the Gram positive bacterium Acidimicrobium ferrooxidans, were selected for detailed analyses. Zinc speciation modeling of the growth media demonstrated that a large fraction of the free metal ion is complexed, potentially affecting its toxicity. Indeed, many of the putative zinc homeostasis genes were constitutively expressed and with the exception of 'F. acidarmanus' ZntA, they were not up-regulated in the presence of excess zinc. Proteomic analysis revealed that zinc played a role in oxidative stress in At. caldus and Am. ferrooxidans. Furthermore, 'F. acidarmanus' kept a constant level of intracellular zinc over all conditions tested whereas the intracellular levels increased with increasing zinc exposure in the remaining organisms.

Antimicrobial resistance is based on the multifarious strategies that bacteria adopt to face antibiotic therapies, making it a key public health concern of our era. Among these strategies, efflux pumps (EPs) contribute significantly to increase the levels and profiles of resistance by expelling a broad range of unrelated compounds - buying time for the organisms to develop specific resistance. In Gram-negative bacteria, many of these chromosomally encoded transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component.One of the strategies to reinvigorate the efficacy of antimicrobials is by joint administration with EP inhibitors (EPI), which either block the substrate binding and/or hinder any of the transport-dependent steps of the pump. In this review, we provide an overview of multidrug-resistance EPs, their inhibition strategies and the relevant findings from the various computational simulation studies reported to date with respect to deciphering the mechanism of action of inhibitors with the purpose of improving their rational design.

The Cl(-) energy gradient across the luminal membrane of Aplysia foregut epithelial cells is directed downhill from the lumen to the cellular cytosol. No primary or secondary active transporters had been shown to be involved in Cl(-) translocation across the luminal membrane. Cl(-) channel blockers impeded the movement of Cl(-) from the lumen into the foregut cellular cytosol. It was concluded that the primary means of Cl(-) transport across the luminal membrane was via Cl(-) conductance.