Pi carrier

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Abstracts

[2] S.Blbulyan, A. Avagyan, A. Poladyan, A.Trchounian. Role of Escherichia coli different hydrogenases in H + efflux and FoF1-ATPase activity during glycerol fermentation at different pH. Bioscience Reports, (2011) 31, 179–184. doi:10.1016/j.bbabio.2014.05.257

S10.P3 Loss of NAD + through the MUC as an additional mechanism of physiological OxPhos uncoupling in Debaryomyces hansenii Alfredo Cabrera-Orefice, Salvador Uribe-Carvajal Instituto de Fisiología Celular, UNAM, Mexico E-mail: [email protected] Debaryomyces hansenii is a salt-tolerant yeast that contains two physiological oxidative phosphorylation (OxPhos) uncoupling mechanisms [1]: a) a mitochondrial unspecific channel (MUC) [2] and b) a branched respiratory chain [3]. In exponential phase grown cells, OxPhos are highly coupled and both uncoupling mechanisms inactive. However, upon aging in culture (stationary growth phase), the efficiency of proton pumping decreases and mitochondria become partially uncoupled, probably in an effort to deplete oxygen in the cell without synthesizing ATP. In the stationary phase, the complex I-dependent rate of oxygen consumption and respiratory coupling are selectively decreased. Complex I activity and expression are not changed in this condition. In fact, all other branched respiratory chain components activities remain the same as in the exponential phase. Uncoupled complex I-dependent respiratory activity is due to lack of NAD +. Here, we propose that NAD + possibly escapes the matrix through an open MUC [4]. When NAD + is added back, coupled complex I-dependent respiratory activity is recovered in both isolated mitochondria and permeabilized spheroplasts [4]. This uptake seems to be catalyzed by a NAD +-specific carrier, which is sensitive to bromocresol purple, bathophenanthroline and pyridoxal5′-phosphate. Loss of matrix NAD + through an open MUC is suggested as a novel OxPhos uncoupling mechanism [4]. References [1] S. Guerrero-Castillo, D. Araiza-Olivera, A. Cabrera-Orefice, J. Espinasa-Jaramillo, M. Gutierrez-Aguilar, L.A. Luevano-Martinez, A. Zepeda-Bastida, S. Uribe-Carvajal, Physiological uncoupling of mitochondrial oxidative phosphorylation. Studies in different yeast species, J. Bioenerg. Biomembr., 43 (2011) 323–331. [2] A. Cabrera-Orefice, S. Guerrero-Castillo, L.A. Luevano-Martinez, A. Pena, S. Uribe-Carvajal, Mitochondria from the salt-tolerant yeast Debaryomyces hansenii (halophilic organelles?), J. Bioenerg. Biomembr., 42 (2010) 11–19. [3] A. Cabrera-Orefice, N. Chiquete-Felix, J. Espinasa-Jaramillo, M. Rosas-Lemus, S. Guerrero-Castillo, A. Pena, S. Uribe-Carvajal, The branched mitochondrial respiratory chain from Debaryomyces hansenii: Components and supramolecular organization, Biochim. Biophys. Acta, 1837 (2014) 73–84. [4] A. Cabrera-Orefice, S. Guerrero-Castillo, R. Diaz-Ruiz, S. UribeCarvajal, Oxidative phosphorylation in Debaryomyces hansenii: Physiological uncoupling at different growth phases, Biochimie, (2014) In Press, Corrected Proof. doi:10.1016/j.bbabio.2014.05.258

S10.P4 Effect of antibody binding on the transport properties of the Na+/H+ Antiporter NhaA from Salmonella Typhimurium

Anika Fippela, Syed Mirb, Christopher Lentesb, Christophe Wirthb, Carola Hunteb a Institute for Biochemistry and Molecular Biology, ZMBZ, Centre for Biological Signalling Studies (BI) b Institute for Biochemistry and Molecular Biology E-mail: anika.fi[email protected] Na+/H+ antiporters are important for ion homoeostasis of bacteria, which have to survive in rapidly changing or adverse environments. These secondary active transporters, which exchange sodium ions against protons, are crucial for control of intracellular pH, cellular Na+ concentration and cell volume in all biological kingdoms of life. X-ray structures of NhaA from Escherichia coli and of NapA from Thermus thermophilus were determined. To obtain further insights in structure/ function relationships and in the mechanism of pH-dependent transport, the Na+/H+ antiporter NhaA from Salmonella enterica serovar Typhimurium LT2 (STNhaA) was crystallized for X-ray structure determination. Following the strategy of antibody fragment mediated crystallization, crystals were obtained with recombinant Fab fragments. Here, we report the characterization of Fab-fragment binding to STNhaA. Purified STNhaA is fully capable of electrogenic Na+/H+ antiport when reconstituted in proteoliposomes. Fluorescence-based transport assays with purified, reconstituted STNhaA:Fab complex clearly showed that transport activity is not impaired by binding of the Fab fragment. Mechanistic implications will be discussed. doi:10.1016/j.bbabio.2014.05.259

S10.P5 Functional and structural characterisation of the human ATP-Mg/Pi carrier Steven Harbornea, Edmund Kunjib a MRC — Mitochondrial Biology Unit, Cambridge, UK b MRC-MBU, UK E-mail: [email protected] The ATP-Mg/Pi carrier (APC) is a member of the mitochondrial carrier family of transport proteins, and carries out the counterexchange of ATP-Mg2 + – for HPO42 - – between the cytosol and the mitochondrial matrix [1–3]. This is the mechanism for net uptake and efflux of adenosine nucleotides in mitochondria [1–3]. The APC has a trans-membrane domain formed of six helices, characteristic of mitochondrial carrier proteins. The APC also has a unique N-terminal extension, which forms a Ca2 + binding regulatory domain [1–3]. The structure of the regulatory domain is known [4], however the mechanism by which calcium regulates transport in the APC is still unresolved. Using biophysical methods such as thermo-stability assays and macromolecular X-ray crystallography the APC has been studied, and new insights about regulation have been gained. References [1] J. Austin & J. R. Aprille, Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria, J. Biol. Chem. 259 (1984) 154–160. [2] G. Fiermonte, F. De Leonardis, S. Todisco, L. Palmieri, F. M. Lasorsa, & F. Palmieri, Identification of the mitochondrial ATP-Mg/Pi transporter: bacterial expression, reconstitution, functional characterization, and tissue distribution, J. Biol. Chem. 279 (2004) 30722–30730. [3] A. del Arco, & J. Satrustegui, Identification of a novel human subfamily of mitochondrial carriers with calcium-binding domains. J. Biol. Chem. 279 (2004) 24701–24713. [4] Q. Yang, S. Brüschweiler & J. J. Chou, A Self-Sequestered Calmodulin-like Ca2 + Sensor of Mitochondrial SCaMC Carrier

Abstracts

and Its Implication to Ca2 + −Dependent ATP-Mg/Pi Transport, Structure, 22 (2014) 1–9. doi:10.1016/j.bbabio.2014.05.260

S10.P6 Do native and mutated forms of huntingtin distinguish human VDAC isoforms? Andonis Karachitosa, Olgierd Stobieniaa, Krzysztof Sobczakb, Vito De Pintoc, Hanna Kmitaa a Laboratory of Bioenergetics, Institute of Molecular Biology and Biotechnology, Faculty of Biology, A, Poland b Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Poland c Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology, University, Poland E-mail: [email protected] The metabolite exchange between mitochondria and cytoplasm is supported by VDAC (voltage-dependent anion selective channel). Studies concerning the role of VDAC have proved that the channel participates in ATP rationing, Ca2 + homeostasis, intracellular redox state regulation, communication between mitochondria and nucleus and apoptosis execution. Thus, the channel is regarded as crucial for mitochondria functioning and consequently for cell life or death. In mitochondria of different organisms VDAC may be present as isoforms encoded by separated genes, displaying different channel-forming activities and probably playing different roles. Saccharomyces cerevisiae mitochondria express two VDAC isoforms (yVDAC1 and yVDAC2), of which only yVDAC1, encoded by POR1 gene, has been proved to form a channel with properties highly conserved in other species. In human mitochondria, as in the case of other vertebrates, three isoforms of VDAC (hVDAC1-hVDAC3) able to form functional channels have been identified. They are expressed in different tissues and organs at different levels. Huntington’s disease (HD) is an autosomal-dominant neurodegenerative hereditary disorder that gradually robs affected individuals of memory, cognitive skills and normal movements. It is originated by the mutation of the gene encoding the huntingtin-protein (Htt). Htt with an abnormal stretch of above 35 glutamines in the N terminus (mHtt) results in HD. The observed symptoms correlate with the selective loss of neurons within the central nervous system, not only in the striatum but also in the cerebral cortex. At present increasing amount of data indicates that mitochondrial functioning is affected by mHtt and the resulting mitochondrial impairments may occur early enough to contribute to mHtt-induced toxicity and the HD pathogenic mechanism. In our studies, we focused on the interaction of hVDAC1hVDAC3 with Htt and mHtt. Therefore, we examined the effect of GSTHtt exon 1 fusion proteins containing 28 (Htt) and 74 (mHtt) glutamines on channel properties of the VDAC proteins isolated from Δpor1hVDAC1, Δpor1hVDAC2 and Δpor1hVDAC3 S. cerevisiae cells as well as from neuroblastoma cells. Obtained results indicate that Htt and mHtt directly and differently modulate human VDAC. This in turn could be important for development of new therapeutic strategies concerning HD. Acknowledgements: the studies were supported by the grant: NCN 2011/01/B/NZ3/00359. doi:10.1016/j.bbabio.2014.05.261

S10.P7 Stochastic model of glycine transporter and its application to amino acid transport in mammalian neurons

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Zaytsev Kirill, Boronovskiy Stanislav, Nartsissov Yaroslav Institute of cytochemistry and molecular pharmacology E-mail: [email protected] Neurotransmitter uptake is quite essential in various metabolic and functional processes in neural tissue. Inhibitory glycinergic neurotransmission is terminated by specific glycine transporters GlyTs (GlyT1 and GlyT2), which actively reuptake glycine from the synaptic cleft. In physiological conditions the pool of this amino acid is regulated by a cascade of metabolic reactions and by membrane transport via specific transporters as well. The re-uptake of glycine into presynaptic terminals and surrounding glia is obligatory for the maintenance of low synaptic levels of the transmitter in the synaptic cleft. Glycine transporter type 2 (GlyT2) presented in the presynaptic membrane is a member of Na +/Cl − -dependent transport proteins family, which share a common structure with 12 transmembrane domains. Transport of one glycine molecule involves the transport of one Cl − and 3 Na + per transport cycle. The main goal of the present study was to develop a computer simulator of GlyT2 activity based on known experimental data for quantitative estimation of membrane glycine transport. A sequence of elemental events happening during the cycle of GlyT2 activity was summarized as a single scheme, which became a basis of an original software. The algorithm of transporter simulator was developed using the probability approach describing the behavior of a single protein. As a result of such computations the number of translocated glycine molecules per time period has been evaluated. The computer experiments were carried out under different environmental conditions such as ion and glycine concentrations. As the major equilibrium constants of the transport steps are still unmeasured the reversibility degree of the glycine transport is also considered as a variable parameter. Using described software the time dependences of glycine, sodium and chloride ions amounts were obtained. Ligand cooperativity was observed for sodium ions (Hill coefficient is 3.6). The developed software based on proposed probability algorithm can be used for a virtual experiment in GLYT2 activity simulation. The described model allows to predict some characteristics of the transporter functioning which can be experimentally proven. This software combined with glycine receptor model can be also used in research laboratories for evaluating concentrations of chloride, sodium and glycine in synaptic cleft and presynaptic terminal in different time points during inhibitory signaling. doi:10.1016/j.bbabio.2014.05.262

S10.P8 The effect of Macrovipera lebetina obtusa viper venom on erythrocyte ghosts membrane ATPase activites Gayane R. Kirakosyan, Hasmik H. Tadevosyan, Narine A. Ghazaryan, Lusine A. Ghulikyan, Naira M. Ayvazyan Orbeli Institute of Physiology, NAS RA, Armenia E-mail: [email protected] Macrovipera lebetina obtusa (MLO) is one of the most important poisonous snakes in Armenia. In the venom of this snake a specific toxin was not identified but they form complexes with other nonenzymatic proteins to achieve higher efficiency through synergy. We have studied influence of venom on the erythrocyte ghosts by fluorescent microscopy. Images were collected on an epi-fluorescent microscope FM320-5M (AmScope, USA). The erythrocyte ghosts were visualized with ANS fluorescent probe. The erythrocyte ghosts were deformed after adding the MLO venom. They shrink within 3 min, and pull in. We also studied activities of Na +, K +-ATPase and Ca2 +-activated Mg2 +-dependent ATPase in the absence and in the presence MLO venom. Venom was added into the assay mixture with low, sub-lethal (0.35 mg/kg approx. 0.5 LD 50 for rat) and lethal