BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

The Mitochondrial Tricarboxylate Carrier of Silver Eel: Chemical Modification by Sulfhydryl Reagents

  • Capobianco, Loredana (Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita di Lecce) ;
  • Impagnatiello, Tecla (Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita di Lecce) ;
  • Ferramosca, Alessandra (Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita di Lecce) ;
  • Zara, Vincenzo (Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita di Lecce)
  • Published : 2004.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

The tricarboxylate (or citrate) carrier was purified from eel liver mitochondria and functionally reconstituted into liposomes. Incubation of the proteoliposomes with various sulfhydryl reagents led to inhibition of the reconstituted citrate transport activity. Preincubation of the proteoliposomes with reversible SH reagents, such as mercurials and methanethiosulfonates, protected the eel liver tricarboxylate carrier against inactivation by the irreversible reagent N-(1-pyrenyl)maleimide (PM). Citrate and L-malate, two substrates of the tricarboxylate carrier, protected the protein against inactivation by sulfhydryl reagents and decreased the fluorescent PM bound to the purified protein. These results suggest that the eel liver tricarboxylate carrier requires a single population of free cysteine(s) in order to manifest catalytic activity. The reactive cysteine(s) is most probably located at or near the substrate binding site of the carrier protein.

Keywords

References

  1. Bisaccia, F., De Palma, A. and Palmieri, F. (1989) Identification and purification of the tricarboxylate carrier from rat liver mitochondria. Biochim. Biophys. Acta 977, 171-176. https://doi.org/10.1016/S0005-2728(89)80068-4
  2. Bisaccia, F., De Palma, A., Prezioso, G. and Palmieri, F. (1990) Kinetic characterization of the reconstituted tricarboxylate carrier from rat liver mitochondria. Biochim. Biophys. Acta 1019, 250-256. https://doi.org/10.1016/0005-2728(90)90201-E
  3. Boyer, P. D. (1954) Spectrophotometric study of the reaction of protein sulfhydryl groups with organic mercurials. J. Am. Chem. Soc. 76, 4331-4337. https://doi.org/10.1021/ja01646a025
  4. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  5. Capobianco, L., Bisaccia, F., Mazzeo, M. and Palmieri, F. (1996) The mitochondrial oxoglutarate carrier: sulfhydryl reagents bind to cysteine-184, and this interaction is enhanced by substrate binding. Biochemistry 35, 8974-8980. https://doi.org/10.1021/bi960258v
  6. Capobianco, L., Ferramosca, A. and Zara, V. (2002) The mitochondrial tricarboxylate carrier of silver eel: dimeric structure and cytosolic exposure of both N- and C-termini. J. Prot. Chem. 21, 515-521. https://doi.org/10.1023/A:1022473504904
  7. Claeys, D. and Azzi, A. (1989) Tricarboxylate carrier of bovine liver mitochondria. Purification and reconstitution. J. Biol. Chem. 264, 14627-14630.
  8. Conover, T. E. (1987) Does citrate transport supply both acetyl groups and NADPH for cytoplasmic fatty acid synthesis? Trends Biochem. Sci. 12, 88-89. https://doi.org/10.1016/0968-0004(87)90042-9
  9. De Pinto, V., Zara, V., Benz, R., Gnoni, G. V. and Palmieri, F. (1991) Characterization of pore-forming activity in liver mitochondria from Anguilla anguilla. Two porins in mitochondria? Biochim. Biophys. Acta 1061, 279-286. https://doi.org/10.1016/0005-2736(91)90293-H
  10. Dierks, T., Salentin, A., Heberger, C. and Kramer, R. (1990a) The mitochondrial aspartate/glutamate and ADP/ATP carrier switch from obligate counterexchange to unidirectional transport after modification by SH-reagents. Biochim. Biophys. Acta 1028, 268-280. https://doi.org/10.1016/0005-2736(90)90176-O
  11. Dierks, T., Salentin, A. and Krämer, R. (1990b) Pore-like and carrier-like properties of the mitochondrial aspartate/glutamate carrier after modification by SH-reagents: evidence for a preformed channel as a structural requirement of carriermediated transport. Biochim. Biophys. Acta 1028, 281-288. https://doi.org/10.1016/0005-2736(90)90177-P
  12. Dulley, J. R. and Grieve, P. A. (1975) A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Anal. Biochem. 64, 136-141. https://doi.org/10.1016/0003-2697(75)90415-7
  13. Dyall, S., Agius, S. C., De Marcos Lousa, C., Trezeguet, V. and Tokatlidis, K. (2003) The dynamic dimerization of the yeast ADP/ATP carrier in the inner mitochondrial membrane is affected by conserved cysteine residues. J. Biol. Chem. 278, 26757-26764. https://doi.org/10.1074/jbc.M302700200
  14. Fiermonte, G., Dolce, V. and Palmieri, F. (1998) Expression in Escherichia coli, functional characterization, and tissue distribution of isoforms A and B of the phosphate carrier from bovine mitochondria. J. Biol. Chem. 273, 22782-22787. https://doi.org/10.1074/jbc.273.35.22782
  15. Hatanaka, T., Kihira, Y., Shinohara, Y., Majima, E. and Terada, H. (2001) Characterization of loops of the yeast mitochondrial ADP/ATP carrier facing the cytosol by site-directed mutagenesis. Biochem. Biophys. Res. Commun. 286, 936-942. https://doi.org/10.1006/bbrc.2001.5498
  16. Hoffmann, B., Stockl, A., Schlame, M., Beyer, K. and Klingenberg, M. (1994) The reconstituted ADP/ATP carrier activity has an absolute requirement for cardiolipin as shown in cysteine mutants. J. Biol. Chem. 269, 1940-1944.
  17. Indiveri, C., Tonazzi, A., Dierks, T., Krämer, R. and Palmieri, F. (1992) The mitochondrial carnitine carrier: characterization of SH-groups relevant for its transport function. Biochim. Biophys. Acta 1140, 53-58. https://doi.org/10.1016/0005-2728(92)90019-X
  18. Indiveri, C., Giangregorio, N., Iacobazzi, V. and Palmieri, F. (2002) Site-directed mutagenesis and chemical modification of the six native cysteine residues of the rat mitochondrial carnitine carrier: implications for the role of cysteine-136. Biochemistry 41, 8649-8656. https://doi.org/10.1021/bi012183n
  19. Kaplan, R. S., Mayor, J. A., Johnston, N. and Oliveira, D. L. (1990) Purification and characterization of the reconstitutively active tricarboxylate transporter from rat liver mitochondria. J. Biol. Chem. 265, 13379-13385.
  20. Kaplan, R. S., Mayor, J. A. and Wood, D. O. (1993) The mitochondrial tricarboxylate transport protein. cDNA cloning, primary structure, and comparison with other mitochondrial transport proteins. J. Biol. Chem. 268, 13682-13690.
  21. Kaplan, R. S., Mayor, J. A., Gremse, D. A. and Wood, D. O. (1995) High level expression and characterization of the mitochondrial citrate transport protein from the yeast Saccharomyces cerevisiae. J. Biol. Chem. 270, 4108-4114. https://doi.org/10.1074/jbc.270.8.4108
  22. Kaplan, R. S., Mayor, J. A., Brauer, D., Kotaria, R., Walters, D. E. and Dean, A. M. (2000a) The yeast mitochondrial citrate transport protein. Probing the secondary structure of transmembrane domain IV and identification of residues that likely comprise a portion of the citrate translocation pathway. J. Biol. Chem. 275, 12009-12016. https://doi.org/10.1074/jbc.275.16.12009
  23. Kaplan, R. S., Mayor, J. A., Kotaria, R., Walters, D. E. and Mchaourab, H. S.(2000b) The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using sitedirected spin labeling. Biochemistry 39, 9157-9163. https://doi.org/10.1021/bi000433e
  24. Kaplan, R. S. (2001) Structure and function of mitochondrial anion transport proteins. J. Membr. Biol. 179, 165-183. https://doi.org/10.1007/s002320010046
  25. Kotaria, R., Mayor, J. A., Walters, D. E. and Kaplan, R. S. (1999) Oligomeric state of wild-type and cysteine-less yeast mitochondrial citrate transport proteins. J. Bioenerg. Biomembr. 31, 543-549. https://doi.org/10.1023/A:1005460810527
  26. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  27. Meijer, A. J. and van Dam, K. (1974) The metabolic significance of anion transport in mitochondria. Biochim. Biophys. Acta 346, 213-244. https://doi.org/10.1016/0304-4173(74)90001-9
  28. Palmieri, F., Stipani, I., Quagliariello, E. and Klingenberg, M. (1972) Kinetic study of the tricarboxylate carrier in rat-liver mitochondria. Eur. J. Biochem. 26, 587-594. https://doi.org/10.1111/j.1432-1033.1972.tb01801.x
  29. Palmieri, F. (1994) Mitochondrial carrier proteins. FEBS Lett. 346, 48-54. https://doi.org/10.1016/0014-5793(94)00329-7
  30. Palmieri, F., Indiveri, C., Bisaccia, F. and Iacobazzi, V. (1995) Mitochondrial metabolite carrier proteins: purification, reconstitution, and transport studies. Methods Enzymol. 260, 349-369. https://doi.org/10.1016/0076-6879(95)60150-3
  31. Palmieri, F., Bisaccia, F., Capobianco, L., Dolce, V., Fiermonte, G., Iacobazzi, V., Indiveri, C. and Palmieri, L. (1996) Mitochondrial metabolite transporters. Biochim. Biophys. Acta 1275, 127-132. https://doi.org/10.1016/0005-2728(96)00062-X
  32. Robinson, B. H., Williams, G. R., Halperin, M. L. and Leznoff, C. C. (1971) Factors affecting the kinetics and equilibrium of exchange reactions of the citrate-transporting system of rat liver mitochondria. J. Biol. Chem. 246, 5280-5286.
  33. Stappen, R. and Kramer, R. (1993) Functional properties of the reconstituted phosphate carrier from bovine heart mitochondria: evidence for asymmetric orientation and characterization of three different transport modes. Biochim. Biophys. Acta 1149, 40-48. https://doi.org/10.1016/0005-2736(93)90022-R
  34. Stipani, I. and Palmieri, F. (1983) Purification of the active mitochondrial tricarboxylate carrier by hydroxylapatite chromatography. FEBS Lett. 161, 269-274. https://doi.org/10.1016/0014-5793(83)81023-0
  35. Tonazzi, A. and Indiveri, C. (2003) Chemical modification of the mitochondrial ornithine/citrulline carrier by SH reagents: effects on the transport activity and transition from carrier to pore-like function. Biochim. Biophys. Acta 1611, 123-130. https://doi.org/10.1016/S0005-2736(03)00033-6
  36. Xu, Y., Mayor, J. A., Gremse, D., Wood, D. O. and Kaplan, R. S. (1995) High-yield bacterial expression, purification, and functional reconstitution of the tricarboxylate transport protein from rat liver mitochondria. Biochem. Biophys. Res. Commun. 207, 783-789. https://doi.org/10.1006/bbrc.1995.1255
  37. Xu, Y., Kakhniashvili, D. A., Gremse, D. A., Wood, D. O., Mayor, J. A., Walters, D. E. and Kaplan, R. S. (2000) The yeast mitochondrial citrate transport protein. Probing the roles of cysteines, Arg(181), and Arg(189) in transporter function. J. Biol. Chem. 275, 7117-7124. https://doi.org/10.1074/jbc.275.10.7117
  38. Wessel, D. and Flügge, U. I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141-143. https://doi.org/10.1016/0003-2697(84)90782-6
  39. Zara, V. and Palmieri, F. (1988) Inhibition and labelling of the mitochondrial 2-oxoglutarate carrier by eosin-5-maleimide. FEBS Lett. 236, 493-496. https://doi.org/10.1016/0014-5793(88)80084-X
  40. Zara, V., Iacobazzi, V., Siculella, L., Gnoni, G. V. and Palmieri, F. (1996) Purification and characterization of the tricarboxylate carrier from eel liver mitochondria. Biochem. Biophys. Res. Commun. 223, 508-513. https://doi.org/10.1006/bbrc.1996.0925
  41. Zara, V., Palmieri, L., Franco, M. R., Perrone, M., Gnoni, G. V. and Palmieri, F. (1998) Kinetics of the reconstituted tricarboxylate carrier from eel liver mitochondria. J. Bioenerg. Biomembr. 30, 555-563. https://doi.org/10.1023/A:1020532500749
  42. Zara, V., Palmieri, L., Giudetti, A., Ferramosca, A., Capobianco, L. and Gnoni, G. V. (2000) The mitochondrial tricarboxylate carrier: unexpected increased activity in starved silver eels. Biochem. Biophys. Res. Commun. 276, 893-898. https://doi.org/10.1006/bbrc.2000.3579
  43. Zara, V., Ferramosca, A. and Palmieri, L. (2003) Structural and functional properties of the mitochondrial metabolite carriers. Recent Res. Devel. Biochem. 4, 255-269.

Cited by

  1. Identification of a novel non-AT1, non-AT2 angiotensin binding site in the rat brain vol.1143, 2007, https://doi.org/10.1016/j.brainres.2007.01.051
  2. Immune system and thiols: Some peculiarities of thiol exchange vol.33, pp.1, 2010, https://doi.org/10.1016/j.cimid.2008.08.001
  3. Abundant expression and purification of biologically active mitochondrial citrate carrier in baculovirus-infected insect cells vol.41, pp.3, 2009, https://doi.org/10.1007/s10863-009-9226-6
  4. Relations Between Structure and Function of the Mitochondrial ADP/ATP Carrier vol.75, pp.1, 2006, https://doi.org/10.1146/annurev.biochem.75.103004.142747
  5. An effective strategy for cloning the mitochondrial citrate carrier: identification, characterization and tissue distribution in silver eel vol.02, pp.03, 2011, https://doi.org/10.4236/abb.2011.23025