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Pyruvate is oxidized to acetyl‐CoA by pyruvate dehydrogenase which is localized in the mitochondrial matrix. The mitochondrial pyruvate carrier (MPC) is composed of SLC54 family members (MPC1 and MPC2) [1, 5], which form functional hetero-dimers [9, 8]. The MPC is expressed in the inner mitochondrial membrane and involved in the import of pyruvate into mitochondria [1, 5]. Ubiquitous disruption of either MPC1 or MPC2 expression results in embryonic lethality [11, 12]. Clinically relevant concentrations of the insulin sensitizers, thiazolidinediones, inhibit the MPC [3]. Other clinically relevant inhibitors of the MPC complex are lonidamine [7, 8], quinolone antibacterials [6], entacapone and nitrofurantoin [8].

Citrin deficiency is a pan-ethnic and highly prevalent mitochondrial disease with three different stages: neonatal intrahepatic cholestasis (NICCD), a relatively mild adaptation stage, and type II citrullinemia in adulthood (CTLN2). The cause is the absence or dysfunction of the calcium-regulated mitochondrial aspartate/glutamate carrier 2 (AGC2/SLC25A13), also called citrin, which imports glutamate into the mitochondrial matrix and exports aspartate to the cytosol. In citrin deficiency, these missing transport steps lead to impairment of the malate-aspartate shuttle, gluconeogenesis, amino acid homeostasis, and the urea cycle. In this review, we describe the geological spread and occurrence of citrin deficiency, the metabolic consequences and use our current knowledge of the structure to predict the impact of the known pathogenic mutations on the calcium-regulatory and transport mechanism of citrin.

The mitochondrial pyruvate carrier (MPC) is critical for cellular homeostasis, as it is required in central metabolism for transporting pyruvate from the cytosol into the mitochondrial matrix. MPC has been implicated in many diseases and is being investigated as a drug target. A few years ago, small membrane proteins, called MPC1 and MPC2 in mammals and Mpc1, Mpc2 and Mpc3 in yeast, were proposed to form large protein complexes responsible for this function. However, the MPC complexes have never been isolated and their composition, oligomeric state and functional properties have not been defined. Here, we identify the functional unit of MPC from Saccharomyces cerevisiae. In contrast to earlier hypotheses, we demonstrate that MPC is a hetero‐dimer, not a multimeric complex. When not engaged in hetero‐dimers, the yeast Mpc proteins can also form homo‐dimers that are, however, inactive. We show that the earlier described substrate transport properties and inhibitor profiles are embodied by the hetero‐dimer. This work provides a foundation for elucidating the structure of the functional complex and the mechanism of substrate transport and inhibition.

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy in transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the Neurotransmitter:Sodium Symporter (NSS) family, Na+ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+ transport. In a step essential for coupled transport, substrate binding must overcome the effect of Na+, allowing intracellular substrate and Na+ release from an inward-open state. However, it is unclear which specific elements of the protein mediate this conformational response to substrate binding. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+ on conformation occurs at the Na2 site, where it influences conformation by fostering interaction between two domains of the protein (JBC 291: 1456, 2016). Here, we identify a conserved tyrosine residue in the substrate binding site required for substrate to enable conversion to inward-open states by establishing an interaction between the two transporter domains. We further identify additional interactions between the two transporter domains in the extracellular pathway that are required. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

Search queries on biomedical databases, such as PubMed, often return a large number of results, only a small subset of which is relevant to the user. Ranking and categorization, which can also be combined, have been proposed to alleviate this information overload problem. Results categorization for biomedical databases is the focus of this work. A natural way to organize biomedical citations is according to their MeSH annotations. MeSH is a comprehensive concept hierarchy used by PubMed. In this paper, we present the BioNav system, a novel search interface that enables the user to navigate large number of query results by organizing them using the MeSH concept hierarchy. First, the query results are organized into a navigation tree. At each node expansion step, BioNav reveals only a small subset of the concept nodes, selected such that the expected user navigation cost is minimized. In contrast, previous works expand the hierarchy in a predefined static manner, without navigation cost modeling. We show that the problem of selecting the best concepts to reveal at each node expansion is NP-complete and propose an efficient heuristic as well as a feasible optimal algorithm for relatively small trees. We show experimentally that BioNav outperforms state-of-the-art categorization systems by up to an order of magnitude, with respect to the user navigation cost. BioNav for the MEDLINE database is available at http://db.cse.buffalo.edu/bionav.

In ion-coupled transport proteins, occupation of selective ion-binding sites is required to trigger conformational changes that lead to substrate translocation. Neurotransmitter transporters, targets of abused and therapeutic drugs, require Na(+) and Cl(-) for function. We recently proposed a chloride-binding site in these proteins not present in Cl(-)-independent prokaryotic homologues. Here we describe conversion of the Cl(-)-independent prokaryotic tryptophan transporter TnaT to a fully functional Cl(-)-dependent form by a single point mutation, D268S. Mutations in TnaT-D268S, in wild type TnaT and in serotonin transporter provide direct evidence for the involvement of each of the proposed residues in Cl(-) coordination. In both SERT and TnaT-D268S, Cl(-) and Na(+) mutually increased each other's potency, consistent with electrostatic interaction through adjacent binding sites. These studies establish the site where Cl(-) binds to trigger conformational change during neurotransmitter transport.

The mitochondrial ADP/ATP carrier imports ADP from the cytosol and exports ATP from the mitochondrial matrix, which are key transport steps for oxidative phosphorylation in eukaryotic organisms. The transport protein belongs to the mitochondrial carrier family, a large transporter family in the inner membrane of mitochondria. It is one of the best studied members of the family and serves as a paradigm for the molecular mechanism of mitochondrial carriers. Structurally, the carrier consists of three homologous domains, each composed of two transmembrane α-helices linked with a loop and short α-helix on the matrix side. The transporter cycles between a cytoplasmic and matrix state in which a central substrate binding site is alternately accessible to these compartments for binding of ADP or ATP. On both the cytoplasmic and matrix side of the carrier are networks consisting of three salt bridges each. In the cytoplasmic state, the matrix salt bridge network is formed and the cytoplasmic network is disrupted, opening the central substrate binding site to the intermembrane space and cytosol, whereas the converse occurs in the matrix state. In the transport cycle, tighter substrate binding in the intermediate states allows the interconversion of conformations by lowering the energy barrier for disruption and formation of these networks, opening and closing the carrier to either side of the membrane in an alternating way. Conversion between cytoplasmic and matrix states might require the simultaneous rotation of three domains around a central translocation pathway, constituting a unique mechanism among transport proteins. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.

BACKGROUND: Gonadotrophin surge-attenuating factor (GnSAF) is an as yet unidentified ovarian factor that acts on the pituitary to attenuate the pre-ovulatory LH surge. In a previous study, GnSAF bioactivity was proposed to derive, at least in part, from a C-terminal domain (95peptide) of human serum albumin (HSA). METHODS AND RESULTS: We employ here the expression-secretion system of Pichia pastoris to produce and assay selected recombinant polypeptides of HSA for GnSAF activity. We show that the C-terminal 95peptide of HSA (residues 490-585; subdomain IIIB) can be expressed from P.pastoris in secreted form and supernatants from clones expressing this polypeptide reduce the GnRH-induced LH secretion of primary rat pituitary cultures by 50-82%. When expressed in the same system, HSA domain III (residues 381-585) or full-length HSA (residues 1-585) are inactive. The bioactive subdomain IIIB is also separable from either domain III or full-length HSA on Blue Sepharose chromatography. CONCLUSIONS: Taken together, the findings highlight the putative importance of HSA subdomain IIIB as a GnSAF-bioactive entity and introduce a unique experimental tool to engineer this molecule for structure-function analysis. Hum Reprod

International audience; Transport proteins of the neurotransmitter sodium symporter (NSS) family regulate the extracellular concentration of several neurotransmitters in the central nervous system. The only member of this family for which atomic-resolution structural data are available is the prokaryotic homologue LeuT. This protein has been used as a model system to study the molecular mechanism of transport of the NSS family. In this Journal Club, we discuss two strikingly different LeuT transport mechanisms: one involving a single high-affinity substrate binding site and one recently proposed alternative involving two high-affinity substrate binding sites that are allosterically coupled.

Serotonin transporter (SERT) is the main target for widely used antidepressant agents. Several of these drugs, including imipramine, citalopram, sertraline, and fluoxetine (Prozac), bound more avidly to SERT in the presence of Cl−. In contrast, Cl−did not enhance cocaine or paroxetine binding. A Cl−binding site recently identified in SERT, and shown to be important for Cl−dependent transport, was also critical for the Cl−dependence of antidepressant affinity. Mutation of the residues contributing to this site eliminated the Cl−-mediated affinity increase for imipramine and fluoxetine. Analysis of ligand docking to a single state of SERT indicated only small differences in the energy of interaction between bound ligands and Cl−. These differences in interaction energy cannot account for the affinity differences observed for Cl−dependence. However, fluoxetine binding led to a conformational change, detected by cysteine accessibility experiments, that was qualitatively different from that induced by cocaine or other ligands. Given the known Cl−requirement for serotonin-induced conformational changes, we propose that Cl−binding facilitates conformational changes required for optimal binding of fluoxetine and other antidepressant drugs.

We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-beta,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose. J Mol Biol

The recent determination of the crystal structure of the leucine transporter from Aquifex aeolicus (aaLeuT) has provided significant insights into the function of neurotransmitter:sodium symporters. Transport by aaLeuT is Cl − independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on Cl − ions. However, the only Cl − ion identified in the aaLeuT structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site is unclear. Here, we use calculations of pK A s and homology modeling to predict the location of a functionally important Cl − binding site in serotonin transporter and other Cl − -dependent transporters. We validate our model through the site-directed mutagenesis of residues predicted to coordinate the Cl − ion and through the observation of sequence conservation patterns in other Cl − -dependent transporters. The proposed site is located midway across the membrane and is formed by residues from transmembrane helices 2, 6, and 7. It is close to the Na1 sodium binding site, thus providing an explanation for the coupling of Cl − and Na + ions during transport. Other implications of the model are also discussed.

We used LeuT, a bacterial homologue of neurotransmitter:sodium symporter family (NSS transporters), as a model system for studying the role of the Na+ ions in NSS mediated transport. LeuT has previously been crystalized in several conformations, providing important insight into the structure of NSS transporters. Two Na+ sites (Na1 and Na2) were observed in LeuT crystal structures. Na1 forms part of the substrate site and Na2 is coordinated by residues from two domains thought to move relative to each other during conformational change. Previous studies using Electron Paramagnetic Resonance (EPR), Forster Resonance Energy Transfer (FRET) and scintillation proximity binding assays have shown that Na+ is required for substrate binding to LeuT and also induces a conformational change which closes the cytoplasmic permeation pathway. This conformational change also opens the extracellular pathway, stabilizing an outward-open LeuT confornation. The molecular mechanism by which LeuT sodium sites affect substrate binding and conformational change has not previously been addressed.We examined the role of each Na+ site on LeuT binding and conformation. Mutation of Na1 (N27D, T254A, N286S) decreased [3H]leucine binding affinity and altered the Na+ dependence of binding. Mutation of Na2 (T354A, S355A) also decreased leucine affinity and dramatically reduced the potency of Na+ for stimulating leucine binding. In detergent-free E.coli membranes from cells expressing LeuT Na+ site mutants, Na+ decreased reactivity of a cysteine replacing Tyr-265 in the cytoplasmic permeation pathway, confirming previous reports that Na+ stabilizes an outward open conformation. Mutation of Na2 dramatically reduced this effect of Na+. Mutation of Na1 also markedly altered the Na+ protection of Cys-265, suggesting that Na1 controls not only leucine affinity but also the conformational equilibrium. These observations were complemented by single molecule FRET measurements and molecular dynamics simulations with the Na1 and Na2 mutants.

Retroviral reverse transcriptase (RT) plays a definite role in retroviral life cycle and is essential for the process of retrotransposition. We investigated the RNA expression of endogenous reverse transcriptases (enRTs) in the NIH3T3 mouse genome using, as a probe, a mixture of RT-PCR generated reverse transcriptase products potentially detecting a large number of RTs following treatment with different agents. We found that the expression of enRTs is induced approximately 500-fold following 5'-azacytidine-treatment. Amongst steroid hormones used such as estradiol, diethylstilbestrol, progesterone and dexamethasone only the latter was effective in inducing enRTs up to 4-fold at a concentration of 10(-7) M. Expression of a mouse dominant-negative form of p53 protein in cell clones resulted in induction of 20- to 50-fold, whereas C2-ceramide in a 4-fold induction at concentrations of 20-80 micro M. In a parallel analysis, the respective expression of the transposable viral-like 30 elements (VL30s) was also measured. Their expression was induced up to 50-fold by 5'-azacytidine, overexpression of the p53 gene and C2-ceramide at 80 micro M. It was also induced approximately 3- to 5-fold following estradiol, diethylstilbestrol or progesterone treatment and 30-fold by dexamethasone. Collectively, our results suggest that such stimuli inducing enRTs might play a role in the activation of transcription and retrotransposition of VL30. Int J Oncol

Prothymosin alpha (ProTalpha) is a histone H1-binding protein that interacts with the transcription coactivator CREB-binding protein and potentiates transcription. Based on coimmunoprecipitation and mammalian two-hybrid assays, we show here that ProTalpha forms a complex with the oncoprotein SET. ProTalpha efficiently decondenses human sperm chromatin, while overexpression of GFP-ProTalpha in mammalian cells results in global chromatin decondensation. These results indicate that decondensation of compacted chromatin fibers is an important step in the mechanism of ProTalpha function.