Homologous and Heterologous Interactions between Hexokinase and Mitochondrial Porin: Evolutionary Implications

Binding of the Type I isozyme of mammalian hexokinase to mitochondria is mediated by the porin present in the outer mitochondrial membrane. Type I hexokinase from rat brain is avidly bound by rat liver mitochondria while, under the same conditions, there is no significant binding to mitochondria from S. cerevisiae. Previously published work demonstrates the lack of significant interaction of yeast hexokinase with mitochondria from either liver or yeast. Thus, structural features required for the interaction of porin and hexokinase must have emerged during evolution of the mammalian forms of these proteins. If these structural features serve no functional role other than facilitating this interaction of hexokinase with mitochondria, it seems likely that they evolved in synchrony since operation of selective pressures on the hexokinase–mitochondrial interaction would require the simultaneous presence of hexokinase and porin capable of at least minimal interaction, and be responsive to changes in either partner that affected this interaction. Recent studies have indicated that a second type of binding site, which may or may not involve porin, is present on mammalian mitochondria. There are also reports of hexokinase binding to mitochondria in plant tissues, but the nature of the binding site remains undefined.     

The specific binding of the microtubule-associated protein 2 (MAP2) to the outer membrane of rat brain mitochondria.

Purified mitochondria from rat brain contain microtubule-associated proteins (MAPs) bound to the outer membrane. Studies of binding in vitro performed with microtubules and with purified microtubule proteins showed that mitochondria preferentially interact with the high-molecular-mass MAPs (and not with Tau protein). Incubation of intact mitochondria with Taxol-stabilized microtubules resulted in the selective trapping of both MAPs 1 and 2 on mitochondria, indicating that an interaction between the two organelles occurred through a site on the arm-like projection of MAPs. Two MAP-binding sites were located on intact mitochondria. The lower-affinity MAP2-binding site (Kd = 2 x 10(-7) M) was preserved and enriched in the outer-membrane fraction, whereas the higher-affinity site (Kd = 1 x 10(-9) M) was destroyed after removing the outer membrane with digitonin. Detergent fractionation of mitochondrial outer membranes saturated with MAP2 bound in vitro showed that MAPs are associated with membrane fragments which contain the pore-forming protein (porin). MAP2 also partially prevents the solubilization of porin from outer membrane, indicating a MAP-induced change in the membrane environment of porin. These observations demonstrate the presence of specific MAP-binding sites on the outer membrane, suggesting an association between porin and the membrane domain involved in the cross-linkage between microtubules and mitochondria.     

Hexokinase-binding properties of the mitochondrial VDAC protein: Inhibition by DCCD and location of putative DCCD-binding sites

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [¹⁴C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257–265 and 275–283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.     

Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary physico-chemical determinants.

The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild-type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild-type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.     

Hexokinase receptor complex in hepatoma mitochondria: evidence from N,N'-dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC

In rapidly growing, highly glycolytic hepatoma cells as much as 65% of the total cell hexokinase is bound to the outer mitochondrial membrane [Parry, D.M., & Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912]. In this paper, we describe the purification to apparent homogeneity of a mitochondrial pore-forming protein from the highly glycolytic AS-30D rat hepatoma cell line. The purified protein shows a single 35 000-dalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an amino acid composition slightly more hydrophobic than that of the rat liver pore protein (also known as VDAC or mitochondrial porin), and a channel-forming activity of 136 channels min-1 (microgram of protein)-1. In addition to displaying the properties characteristic of VDAC (single-channel conductance, voltage dependence, and preference for anions), we observe that the AS-30D VDAC protein is one of only three mitochondrial proteins that bind [14C]dicyclohexylcarbodiimide (DCCD) at relatively low dosages (2 nmol of DCCD/mg of mitochondrial protein). Significantly, treatment of intact mitochondria isolated from either rat liver or the AS-30D hepatoma with DCCD results in an almost complete inhibition of their ability to binding hexokinase. Fifty percent inhibition of binding occurs at less than 2 nmol of DCCD/mg of mitochondrial protein. In contrast to DCCD, water-soluble carbodiimides are without effect on hexokinase binding. These results suggest that the pore-forming protein of tumor mitochondria forms at least part of the hexokinase receptor complex. In addition, they indicate that a carboxyl residue located within a hydrophobic region of the receptor complex may play a critical role in hexokinase binding.     

VDAC and peripheral channelling complexes in health and disease

VDAC changes its structure either voltage dependent in artificial membranes or physiologically by interaction with the c conformation of the adenine nucleotide translocator (ANT). This interaction creates contact sites and leads to a specific organisation of cytochrome c in the VDAC ANT complexes. The VDAC structure specific for contact sites thus generates a signal at the surface for several proteins in the cytosol to bind with high affinity such as hexokinase, glycerolkinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC ANT complex has two critical qualities: firstly, external Bax gets access to the cytochrome c and secondly the ANT stays in the c conformation that easily changes the structure to an unspecific uni-porter causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as specific anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly hinders direct interaction between VDAC and ANT and secondly changes porin structure into low affinity for hexokinase and external Bax. Cytochrome c in the creatine kinase complex will be differently organised not accessible to Bax and the ANT is run as anti-porter by the active octamer. However, when free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax dependent cytochrome c release and risk of permeability transition pore opening.     

Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin

Mitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria.     

The role of the mitochondrial outer membrane in energy metabolism of tumor cells

The outer mitochondrial membrane contains a pore structure which is composed of a 30,000 Da protein, porin. The pore has an internal diameter of 2 nm and exhibits a molecular-sieving exclusion limit between 3000 and 6000 Da. These pores, therefore, provide the exit/entrance port for metabolites moving between mitochondria and the cytosol. Hexokinase binds to porin on the outer surface of mitochondria. The location of hexokinase has evoked a number of theories in which bound hexokinase is given a central role in regulating glycolysis, and, perhaps, the metabolic communication between oxidative and glycolytic metabolism. This is of particular importance in rapidly growing tumor cells in which the aerobic production of lactate and hexokinase activity are highly induced. In the present paper, we summarize the suggested roles of the outer membrane and bound hexokinase in regulation glycolysis of tumor cells. Experiments attempting to elucidate the role of hexokinase binding in the regulation of tumor cell metabolism are presented.     

Mitochondrial boundary membrane contact sites in brain: points of hexokinase and creatine kinase location, and control of Ca2+ transport

The location of hexokinase at the surface of brain mitochondria was investigated by electron microscopy using immuno-gold labelling techniques. The enzyme was located where the two mitochondrial limiting membranes were opposed and contact sites were possible. Disruption of the outer membrane by digitonin did not remove bound hexokinase and creatine kinase from brain mitochondria, although the activity of outer membrane markers and adenylate kinase decreased, suggesting a preferential location of both enzymes in the contact sites. In agreement with that, a membrane fraction was isolated from osmotically lysed rat brain mitochondria in which hexokinase and creatine kinase were concentrated. The density of this kinase-rich fraction was specifically increased by immuno-gold labelling of hexokinase, allowing a further purification by density gradient centrifugation. The fraction was composed of inner and outer limiting membrane components as shown by the specific marker enzymes, succinate dehydrogenase and NADH-cytochrome-c-oxidase (rotenone insensitive). As reported earlier for the enriched contact site fraction of liver mitochondria the fraction from brain mitochondria contained a high activity of glutathione transferase and a low cholesterol concentration. Moreover, the contacts showed a higher Ca2+ binding capacity in comparison to outer and inner membrane fractions. This finding may have regulatory implications because glucose phosphorylation via hexokinase activated the active Ca2+ uptake system and inhibited the passive efflux, resulting in an increase of intramitochondrial Ca2+.     

Binding of non-catalytic ATP to human hexokinase I highlights the structural components for enzyme-membrane association control

BACKGROUND: Hexokinase I sets the pace of glycolysis in the brain, catalyzing the ATP-dependent phosphorylation of glucose. The catalytic properties of hexokinase I are dependent on product inhibition as well as on the action of phosphate. In vivo, a large fraction of hexokinase I is bound to the mitochondrial outer membrane, where the enzyme adopts a tetrameric assembly. The mitochondrion-bound hexokinase I is believed to optimize the ATP/ADP exchange between glucose phosphorylation and the mitochondrial oxidative phosphorylation reactions. RESULTS: The crystal structure of human hexokinase I has been determined at 2.25 A resolution. The overall structure of the enzyme is in keeping with the closed conformation previously observed in yeast hexokinase. One molecule of the ATP analogue AMP-PNP is bound to each N-terminal domain of the dimeric enzyme in a surface cleft, showing specific interactions with the nucleotide, and localized positive electrostatic potential. The molecular symmetry brings the two bound AMP-PNP molecules, at the centre of two extended surface regions, to a common side of the dimeric hexokinase I molecule. CONCLUSIONS: The binding of AMP-PNP to a protein site separated from the catalytic centre of human hexokinase I can be related to the role played by some nucleotides in dissociating the enzyme from the mitochondrial membrane, and helps in defining the molecular regions of hexokinase I that are expected to be in contact with the mitochondrion. The structural information presented here is in keeping with monoclonal antibody mapping of the free and mitochondrion-bound forms of the enzyme, and with sequence analysis of hexokinases that differ in their mitochondria binding properties.     

Interaction of hexokinase with the outer mitochondrial membrane and a hydrophobic matrix

The major portion of rat brain hexokinase (HK type I) is bound to the outer membrane of mitochondria and glucose-6-phosphate (G6P) can release the bound enzyme. In an attempt to look at the hydrophobic component of binding, interaction of the enzyme with a purely hydrophobic matrix, palmityl-substituted Sepharose-4B (Sepharose-lipid) was investigated. Hexokinase readily bound to this matrix with retention of its catalytic activity. Glucose-6-phosphate which has a releasing effect on the mitochondrially bound enzyme, enhanced binding of the enzyme on the hydrophobic matrix. Chymotrypsin treatment of hexokinase which causes loss of binding to mitochondria, also results in loss of adsorption to the hydrophobic matrix, thus demonstrating that the hydrophobic tail present at its N-terminal end is essential for binding in both cases. Data presented provide some new information relevant to understanding how hexokinase interacts with its natural binding matrix, the mitochondrion.     

Hexokinase ‘binding’ of normal and tumoral human brain mitochondria

Interaction of type I hexokinase (HK-I) with the mitochondria obtained from the biopsy specimens of normal and tumoral human brain tissues was studied in the present investigation. This effort was undertaken with the aim of exploring possible differences in the mode of association of the enzyme with the outer mitochondrial membrane in the described sources. Results indicate that the two sites for binding of HK-I suggested in the literature, based on extensive studies carried out on rat brain mitochondria, are similarly present in the human brain mitochondria. Differences in the microenvironments of HK binding, as reflected by the presented data, are suggested to be of importance in regulation of the catalytic potential of the bound enzyme. The real metabolic significance of this association in relation to cancer and its practical importance would need further investigation.     

Regulation of hexokinase binding to VDAC

Hexokinase isoforms I and II bind to mitochondrial outer membranes in large part by interacting with the outer membrane voltage-dependent anion channel (VDAC). This interaction results in a shift in the susceptibility of mitochondria to pro-apoptotic signals that are mediated through Bcl2-family proteins. The upregulation of hexokinase II expression in tumor cells is thought to provide both a metabolic benefit and an apoptosis suppressive capacity that gives the cell a growth advantage and increases its resistance to chemotherapy. However, the mechanisms responsible for the anti-apoptotic effect of hexokinase binding and its regulation remain poorly understood. We hypothesize that hexokinase competes with Bcl2 family proteins for binding to VDAC to influence the balance of pro-and anti-apoptotic proteins that control outer membrane permeabilization. Hexokinase binding to VDAC is regulated by protein kinases, notably glycogen synthase kinase (GSK)-3β and protein kinase C (PKC)-ɛ. In addition, there is evidence that the cholesterol content of the mitochondrial membranes may contribute to the regulation of hexokinase binding. At the same time, VDAC associated proteins are critically involved in the regulation of cholesterol uptake. A better characterization of these regulatory processes is required to elucidate the role of hexokinases in normal tissue function and to apply these insights for optimizing cancer treatment.     

Role of phosphorylation of porin (VDAC) in regulation of mitochondrial outer membrane under normal conditions and alcohol intoxication

The present work is an overview of the factors regulating permeability of the outer membrane of mitochondria and the state of the channels formed by porin (voltage-dependent anion channels, VDAC). According to the accumulated data, modulation of the outer membrane permeability can be induced by endogenous phosphorylation of VDAC channels. Different protein kinases, such as protein kinase A, protein kinase C, tyrosine protein kinase, hexokinase, glycogen synthetase kinase-3β (GSK-3β), Akt and p38 kinases, were shown to be involved in VDAC phosphorylation. Among these protein kinases, alcohol-induced stress-kinases, GSK-3β, Akt, and p38 identified in mitochondria may participate in phosphorylation of porin, modulation of VDAC conductance, and regulation of the outer membrane permeability.     

Intracellular distribution of hexokinase in rabbit brain

Hexokinase in mammalian brain is particulate and usually considered to be bound to the outer mitochondrial membrane. Investigation of rabbit brain mitochondria prepared either by differential centrifugation and discontinuous density gradient centrifugation has provided evidence that this particulate fraction also contains endoplasmic vesicles and synaptosomes. Solubilization of the bound hexokinase by different combinations of detergents and metabolites has proved the existence of different hexokinase binding sites. Electron microscopic examination of hexokinase location by immuno-gold labelling techniques confirmed, that hexokinase is indeed predominantly bound to mitochondria but that a significant proportion is also bound to non-mitochondrial membranes. Attempts to quantify this distribution were unsuccessful since different figures were obtained using anti-hexokinase IgG affinity purified on immobilized native or denatured hexokinase. Binding studies of the purified rabbit brain mitochondrial hexokinase to rabbit liver mitochondria and microsomes confirmed that in addition to a binding site on mitochondria there is another binding site on microsomes. The N-terminal sequence of hexokinase has been shown to be important for mitochondria binding and also for microsome binding. These results suggest that the intracellular localization of hexokinase in rabbit brain is not exclusively mitochondrial and that the metabolic role of this enzyme should be reconsidered by including a binding site on the endoplasmic reticulum.     

The function of complexes between the outer mitochondrial membrane pore (VDAC) and the adenine nucleotide translocase in regulation of energy metabolism and apoptosis

The outer mitochondrial membrane pore (VDAC) changes its structure either voltage-dependently in artificial membranes or physiologically by interaction with the adenine nucleotide translocase (ANT) in the c-conformation. This interaction creates contact sites and leads in addition to a specific organisation of cytochrome c in the VDAC-ANT complexes. The VDAC structure that is specific for contact sites generates a signal at the surface for several proteins in the cytosol to bind with high capacity, such as hexokinase, glycerol kinase and Bax. If the VDAC binding site is not occupied by hexokinase, the VDAC-ANT complex has two critical qualities: firstly, Bax gets access to cytochrome c and secondly the ANT is set in its c-conformation that easily changes conformation into an unspecific channel (uniporter) causing permeability transition. Activity of bound hexokinase protects against both, it hinders Bax binding and employs the ANT as anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC from the inner surface of the outer membrane. This firstly restrains interaction between VDAC and ANT and secondly changes the VDAC structure into low affinity for hexokinase and Bax. Cytochrome c in the creatine kinase complex will be differently organised, not accessible to Bax and the ANT is run as anti-porter by the active creatine kinase octamer. However, when, for example, free radicals cause dissociation of the octamer, VDAC interacts with the ANT with the same results as described above: Bax-dependent cytochrome c release and risk of permeability transition pore opening.     

Cross-linking analysis of yeast mitochondrial outer membrane

By enrichment of contact sites between the two mitochondrial boundary membranes it has been shown that this fraction contained a high activity of glutathione transferase and hexokinase which was bound to the outer membrane pore protein (Ohlendieck, K. et al. (1986) Biochim. Biophys. Acta 860, 672-689). Therefore, an interaction between the three proteins in the contact sites has been suggested. Cross-linking experiments with isolated outer membrane of yeast mitochondria show that glutathione transferase and the pore protein are already associated in the free outer membrane. Porin appeared to adopt four different oligomeric complexes in the membrane, including interactions with a 14 kDa polypeptide, which has glutathione transferase activity. The latter polypeptide could be phosphorylated by intrinsic or extrinsic protein kinases, while the porin itself was not phosphorylated. Yeast hexokinase, when bound to the outer membrane, was able to cross-link to the pore protein.     

Microcompartmentation of energy metabolism at the outer mitochondrial membrane: Role in diabetes mellitus and other diseases

Complexes made up of the kinases, hexokinase and glycerol kinase, together with the outer mitochondrial membrane voltage-dependent anion channel (VDAC) protein, porin, and the inner mitochondrial membrane protein, the adenine nucleotide translocator, are involved in tumorigenesis, diabetes mellitus, and central nervous system function. Identification of these two mitochondrial membrane proteins, along with an 18 kD protein, as components of the peripheral benzodiazepine receptor, provides independent confirmation of the interaction of porin and the adenine nucleotide translocator to form functional contact sites between the inner and outer mitochondrial membranes. We suggest that these are dynamic structures, with channel conductances altered by the presence of ATP, and that ligand-mediated conformational changes in the porin-adenine nucleotide translocator complexes may be a general mechanism in signal transduction.     

Integration of porin synthesized in vitro into outer mitochondrial membranes

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).