Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris)

Abstract

Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S–13S) and large (>40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Abbreviations

DDM:

Dodecylmaltoside

HK:

Hexokinase

LC–MS/MS:

Liquid chromatography coupled to tandem mass spectrometry

MS:

Mass spectrometry

OMM:

Outer mitochondrial membrane(s)

PK:

Pyruvate kinase

PMSF:

Phenylmethylsulfonyl fluoride

SUS:

Sucrose synthase

VDAC:

Voltage-dependent anion channel (mitochondrial porin)

References

  1. Angeles-Nunez JG, Tiessen A (2010) Arabidopsis sucrose synthase 2 and 3 modulate metabolic homeostasis and direct carbon towards starch synthesis in developing seeds. Planta 232:701–718

  2. Arora KK, Pedersen PL (1988) Functional significance of mitochondrial bound hexokinase in tumor cell metabolism. Evidence for preferential phosphorylation of glucose by intramitochondrially generated ATP. J Biol Chem 263:17422–17428

  3. Balasubramanian R, Karve A, Kandasamy M, Meagher RB, Moore Bd (2007) A role for F-actin in hexokinase-mediated glucose signaling. Plant Physiol 145:1423–1434

  4. BeltrandelRio H, Wilson JE (1992) Interaction of mitochondrially bound rat brain hexokinase with intramitochondrial compartments of ATP generated by oxidative phosphorylation and creatine kinase. Arch Biochem Biophys 299:116–124

  5. Camacho-Pereira J, Meyer LE, Bender ML, Fernandes OM, Galina A (2009) Reactive oxygen species production by potato tuber mitochondria is modulated by mitochondrially bound hexokinase activity. Plant Physiol 149:1099–1110

  6. Claeyssen E, Rivoal J (2007) Isozymes of plant hexokinase: occurrence, properties and functions. Phytochemistry 68:709–731

  7. Damari-Weissler H, Ginzburg A, Gidoni D, Mett A, Krassovskaya I, Weber APM, Belausov E, Granot D (2007) Spinach SoHXK1 is a mitochondria-associated hexokinase. Planta 226:1053–1058

  8. da-Silva WS, Rezende GL, Galina A (2001) Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation. J Exp Bot 52:1191–1201

  9. Delmer DP (1999) Cellulose biosynthesis: exciting times for a difficult field of study. Annu Rev Plant Physiol Plant Mol Biol 50:245–276

  10. Douce R, Bourguignon J, Brouquisse R, Neuburger M (1987) Isolation of plant mitochondria: general principles and criteria of integrity. Methods Enzymol 148:403–415

  11. Duncan O, Taylor NL, Carrie C, Eubel H, Kubiszewski-Jakubiak S, Zhang B, Narsai AR, Millar H, Whelan J (2011) Multiple lines of evidence localize signaling, morphology, and lipid biosynthesis machinery to the mitochondrial outer membrane of Arabidopsis. Plant Physiol 157:1093–1113

  12. Fiek C, Benz R, Roos N, Brdiczka D (1982) Evidence for identity between the hexokinase-binding protein and the mitochondrial porin in the outer membrane of rat liver mitochondria. Biochim Biophys Acta 688:429–440

  13. Florchinger M, Zimmermann M, Traub M, Neuhaus HE, Mohlmann T (2006) Adenosine stimulates anabolic metabolism in developing castor bean (Ricinus communis L.) cotyledons. Planta 223:340–348

  14. Galina A, da Silva WS (2000) Hexokinase activity alters sugar-nucleotide formation in maize root homogenates. Phytochemistry 53:29–37

  15. Galina A, Reis M, Albuquerque MC, Gómez Puyou A, Gomez Puyou MT, de Meis L (1995) Different properties of the mitochondrial and cytosolic hexokinases in maize roots. Biochem J 309:105–112

  16. Giegé P, Heazlewood JL, Ressner-Tunali U, Millar H, Fernie AR, Leaver CJ, Sweetlove LJ (2003) Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells. Plant Cell 15:2140–2151

  17. Goldin N, Arzoine L, Heyfets A, Israelson A, Zaslavsky Z, Bravman T, Bronner V, Notcovich A, Shoshan-Barmatz V, Flescher E (2008) Methyl jasmonate binds to and detaches mitochondria-bound hexokinase. Oncogene 27:4636–4643

  18. González de la Vara LE, Lino Alfaro M (2009) Separation of membrane proteins according to their hydropathy by serial phase partitioning with Triton X-114. Anal Biochem 387:280–286

  19. González de la Vara LE, Medina G (1990) Phosphorylation by inorganic phosphate of the plasma membrane H+-ATPase from red beet (Beta vulgaris L.). Plant Physiol 94:1522–1527

  20. Granot D (2008) Putting plant hexokinases in their proper place. Phytochemistry 69:2649–2654

  21. Heldt HW (2005) Plant biochemistry, 3rd edn. Elsevier Academic Press, London. ISBN: 0-12-088391-033

  22. Hoogenboom BW, Suda K, Engel A, Fotiadis D (2007) The supramolecular assemblies of voltage-dependent anion channels in the native membrane. J Mol Biol 370:246–255

  23. Karve A, Rauh BL, Xia X, Kandasamy M, Meagher RB, Sheen J, Moore Bd (2008) Expression and evolutionary features of the hexokinase gene family in Arabidopsis. Planta 228:411–425

  24. Karve R, Lauria M, Virnig A, Xia X, Rauh BL, Moore Bd (2010) Evolutionary lineages and functional diversification of plant hexokinases. Mol Plant 3:334–346

  25. Klodmann J, Senkler M, Rode C, Braun HP (2011) Defining the protein complex proteome of plant mitochondria. Plant Physiol 157:587–598

  26. Koch K (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr Opin Plant Biol 7:235–246

  27. Linden M, Gellerfors P, Nelson BD (1982) Pore protein and the hexokinase-binding protein from the outer membrane of rat liver mitochondria are identical. FEBS Lett 141:189–192

  28. Lino B, Carrillo-Rayas MT, Chagolla A, González de la Vara LE (2006) Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes. Planta 225:255–268

  29. Loef I, Stitt M, Geigenberger P (1999) Orotate leads to a specific increase in uridine nucleotide levels and a stimulation of sucrose degradation and starch synthesis in discs from growing potato tubers. Planta 209:314–323

  30. Lunn JE (2007) Compartmentation in plant metabolism. J Exp Bot 58:35–47

  31. Mannella CA (1987) Isolation of the outer membrane of plant mitochondria. Methods Enzymol 148:453–464

  32. Miernyk JA, Dennis DT (1983) Mitochondrial, plastid, and cytosolic isozymes of hexokinase from developing endosperm of Ricinus communis. Arch Biochem Biophys 226:458–468

  33. Nakashima RA, Mangan PS, Colombini M, Pedersen PL (1986) Hexokinase receptor complex in hepatoma mitochondria: evidence from N,N′-dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC. Biochemistry 25:1015–1021

  34. Nilsson A, Olsson T, Ulfstedt M, Thelander M, Ronne H (2011) Two novel types of hexokinases in the moss Physcomitrella patens. BMC Plant Biol 11:32

  35. Pastorino JG, Hoek JB (2008) Regulation of hexokinase binding to VDAC. J Bioenerg Biomembr 40:171–182

  36. Pedersen PL (2008) Voltage dependent anion channels (VDACs): a brief introduction with a focus on the outer mitochondrial compartment’s roles together with hexokinase-2 in the “Warburg effect” in cancer. J Bioenerg Biomembr 40:123–126

  37. Petraglia T, Poole RJ (1980) Effect of anoxia on ATP levels and ion transport rates in red beet. Plant Physiol 65:973–974

  38. Rezende GL, Logullo C, Meyer L, Machado LB, Oliveira-Carvalho AL, Zingali RB, Cifuentes D, Galina A (2006) Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes. Braz J Med Biol Res 39:1159–1169

  39. Rosano C (2011) Molecular model of hexokinase binding to the outer mitochondrial membrane porin (VDAC1): implication for the design of new cancer therapies. Mitochondrion 11:513–519

  40. Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

  41. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858

  42. Shevchenko A, Sunyaev S, Loboda A, Shevchenko A, Bork P, Ens W, Standing KG (2001) Charting the proteomes of organisms with unsequenced genomes by MALDI-quadrupole time-of-flight mass spectrometry and BLAST homology searching. Anal Chem 73:1917–1926

  43. Stoschek CM (1990) Increased uniformity in the response of the coomassie blue G protein assay to different proteins. Anal Biochem 184:111–116

  44. Tiessen A, Hendriks JHM, Stitt M, Branscheid A, Gibon Y, Farre EM, Geigernberger P (2002) Starch synthesis in potato tubers is regulated by post-translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to the sucrose supply. Plant Cell 14:2191–2213

  45. Tiessen A, Nerlich A, Faix B, Hümmer C, Fox S, Trafford K, Weber H, Weschke W, Geigernberger P (2012) Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method. J Exp Bot 63:2071–2087

  46. Vanoni MA, Curti B (2008) Structure–function studies of glutamate synthases: a class of self-regulated iron–sulfur flavoenzymes essential for nitrogen assimilation. IUBMB Life 60:287–300

  47. Wilson JE (2003) Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function. J Exp Biol 206:2049–2057

  48. Wittig I, Braun H-P, Schägger H (2006) Blue native PAGE. Nat Protoc 1:418–428

  49. Xie GC, Wilson JE (1988) Rat brain hexokinase: the hydrophobic N-terminus of the mitochondrially bound enzyme is inserted in the lipid bilayer. Arch Biochem Biophys 267:803–810

  50. Young BD (1984) Measurement of sedimentation coefficients and computer simulation of rate-zonal separations. In: Rickwood D (ed) Centrifugation. A practical approach, 2nd edn. IRL Press, Oxford, pp 127–155. ISBN: 0-9004147-55X

Download references

Acknowledgments

We thank Bárbara Lino, Marisol Piceno and Rocío Medina for their help. This work was supported by Consejo Nacional de Ciencia y Tecnología (Conacyt, Mexico) by granting a postgraduate fellowship to FC Alcántar and funding basic-science projects conducted by L. González, J.P. Delano and A. Tiessen.

Author information

Correspondence to Luis Eugenio González de la Vara.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online resource 1 (PDF 331 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Alcántar-Aguirre, F.C., Chagolla, A., Tiessen, A. et al. ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris). Planta 237, 1571–1583 (2013). https://doi.org/10.1007/s00425-013-1866-4

Download citation

Keywords

  • Metabolic channeling
  • Energy metabolism
  • Outer mitochondrial membrane proteins
  • Plant mitochondria