Journal of Bioenergetics and Biomembranes

, Volume 31, Issue 6, pp 569–579 | Cite as

Binding of Rat Brain Hexokinase to Recombinant Yeast Mitochondria: Identification of Necessary Molecular Determinants

Article

Abstract

The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wildtype, porinless, or expressing recombinant human porin) was studied in an effort to identifyminimal requirements for each component. A short hydrophobic N-terminal peptide ofhexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria.Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loopsaround residues 110 and 200, as determined by proteolytic-fragment identification usingantibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysisthan wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboringseveral natural or engineered porins from various sources, bind hexokinase to variable extentwith marked preference for the mammalian porin1 isoform. Genetic alteration of this isoformat the C-, but not the N-terminal, results in a significant reduction of hexokinase bindingability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme toall recombinant mitochondria, as well as to proteolytically digested organelles. Consequently,brain hexokinase association with heterologous mitochondria (yeast) in these conditions occursto an extent comparable to that with homologous (rat) mitochondria. The study, also pertinentto the topology and organization of porin in the membrane, represents a necessary first stepin the functional investigation of the physiological role of mammalian hexokinase binding tomitochondria in reconstituted heterologous recombinant systems, as models to cellularmetabolism.

Heterologous expression mitochondrial porin VDAC topology hexokinase binding macromolecular recognition cellular organization 

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REFERENCES

  1. Aflalo, C., and Azoulay, H. (1998). J. Bioenerg. Biomembr. 30, 245-255.Google Scholar
  2. Aleshin, A. E., Fromm, H. J., and Honzatko, R. B. (1998). FEBS Lett. 434, 42-46.Google Scholar
  3. Arora, K. K., and Pedersen, P. L. (1988). J. Biol. Chem. 263, 17422-17428.Google Scholar
  4. Arora, K. K., Filburn, C. R., and Pedersen, P. L. (1993). J. Biol. Chem. 268, 18259-18266.Google Scholar
  5. Aubert-Foucher, E., Font, B., and Gautheron, D. C. (1984). Arch. Biochem. Biophys. 232, 391-399.Google Scholar
  6. Azoulay, H., and Aflalo, C. (1996). In BioThermoKinetics of the Living Cell(Westerhoff, H. V., Snoep, J. L., Sluse, F. E., Wijker, J. E., and Kholodenko, B. N., eds.), BioThermoKinetics Press, Amsterdam, pp. 289-294.Google Scholar
  7. Babel, D., Walter, G., Gotz, H., Thinnes, F. P., Jurgens, L., Konig, U., and Hilschmann, N. (1991). Hoppe Seyler Biol. Chem. 372, 1027-1034.Google Scholar
  8. Benz, R. (1994). Biochim. Biophys. Acta 1197, 167-196.Google Scholar
  9. Beutner, G., Ruck, A., Riede, B., and Brdiczka, D. (1998). Biochim. Biophys. Acta 1368, 7-18.Google Scholar
  10. Blachly-Dyson, E., Peng, S., Colombini, M., and Forte, M. (1990). Science 247, 1233-1236.Google Scholar
  11. Blachly-Dyson, E., Zambronicz, E. B., Yu, W. H., Adams, V., McCabe, E. R., Adelman, J., Colombini, M., and Forte, M. (1993). J. Biol. Chem. 268, 1835-1841.Google Scholar
  12. Blachly-Dyson, E., Song, J., Wolfgang, W. J., Colombini, M., and Forte, M. (1997). Mol. Cell. Biol. 17, 5727-5738.Google Scholar
  13. Bustamente, E., Morris, H. P., and Petersen, P. L. (1981). J. Biol. Chem. 256, 8699-8707.Google Scholar
  14. Cesar, M. D., and Wilson, J. E. (1998). Arch. Biochem. Biophys. 350, 109-117.Google Scholar
  15. De Pinto, V., Prezioso, G., Thinnes, F., Link, T. A., and Palmieri, F. (1991). Biochemistry 30, 10191-10200.Google Scholar
  16. Elkeles, A., Devos, K., Grauer, D., Zizi, M., and Breiman, A. (1995). Plant Mol. Biol 29, 109-124.Google Scholar
  17. Elkeles, A., Breiman, A., and Zizi, M. (1997). J. Biol. Chem. 272, 6252-6260.Google Scholar
  18. Felgner, P. L., Messer, J. L., and Wilson, J. E. (1979). J. Biol. Chem. 254, 4946-4949.Google Scholar
  19. Gelb, B. D., Adams, V., Jones, S. N., Griffin, L. D., MacGregor, G. R., and McCabe, E. R. (1992). Proc. Natl. Acad. Sci. USA 89, 202-206.Google Scholar
  20. Laemmli, U. K. (1970). Nature London 227, 680-685.Google Scholar
  21. Laterveer, F. D., Gellerich, F. N., and Nicolay, K. (1995). Eur. J. Biochem. 232, 569-577.Google Scholar
  22. Magnani, M., Crinelli, R., Antonelli, A., Casabianca, A., and Serafini, G. (1994). Biochim. Biophys. Acta 1206, 180-190.Google Scholar
  23. Mannella, C. A. (1997). J. Bioenerg. Biomebr. 29, 525-531.Google Scholar
  24. Mihara, K., Blobel, G., and Sato, R. (1982). Proc. Natl. Acad. Sci. USA 79, 7102-7106.Google Scholar
  25. Minton, A. (1993). J. Mol. Recognition 6, 211-214.Google Scholar
  26. Mulichak, A., Wilson, J., Padmanabhan, K., and Garavito, R. (1998). Nature Struct. Biol. 5, 555-560.Google Scholar
  27. Polakis, P. G., and Wilson, J. E. (1985). Arch. Biochem. Biophys. 236, 328-337.Google Scholar
  28. Sampson, M. J., Lovell, R. S., and Craigen, W. J. (1997). J. Biol. Chem. 272, 18966-18973.Google Scholar
  29. Shafir, I., Feng, W., and Shoshan-Barmatz, V. (1998). Eur. J. Biochem. 253, 627-636.Google Scholar
  30. Song, J., and Colombini, M. (1996). J. Bioenerg. Biomembr. 28, 153-161.Google Scholar
  31. Stocchi, V., Fiorani, M., Biagiarelli, B., Piccoli, G., Saltarelli, R., Palma, F., Cucchiarini, L., and Dacha, M. (1995). Biochem. Mol. Biol. Intern. 35, 1133-1142.Google Scholar
  32. Sui, D., and Wilson, J. E. (1997). Arch. Biochem. Biophys. 345, 111-125.Google Scholar
  33. Towbin, J. A., Minter, M., Brdiczka, D., Adams, V., de Pinto, V., Palmieri, F., and McCabe, E. R. (1989). Biochem. Med. Metab. Biol. 42, 161-169.Google Scholar
  34. Wicker, U., Bucheler, K., Gellerich, F. N., Wagner, M., Kapischke, M., and Brdiczka, D. (1993). Biochim. Biophys. Acta 1142, 228-239.Google Scholar
  35. Wilson, J. (1978). Trends Biochem. Sci. 3, 124-125.Google Scholar
  36. Wilson, J. E. (1995). Rev. Physiol. Biochem. Pharmacol. 126, 65-198.Google Scholar
  37. Wilson, J. E. (1997). J. Bioenerg. Biomembr. 29, 97-102.Google Scholar
  38. Wilson, J. E., and Smith, A. D. (1985). J. Biol. Chem. 260, 12838-12843.Google Scholar
  39. Xie, G., and Wilson, J. E. (1990). Arch. Biochem. Biophys. 276, 285-293.Google Scholar
  40. Xie, G. C., and Wilson, J. E. (1988). Arch. Biochem. Biophys. 267, 803-810.Google Scholar
  41. Yu, W. H., Wolfgang, W., and Forte, M. (1995). J. Biol. Chem. 270, 13998-14006.Google Scholar

Copyright information

© Plenum Publishing Corporation 1999

Authors and Affiliations

  1. 1.Department of Life SciencesBen Gurion University of the NegevBeer ShevaIsrael

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