VDAC1 serves as a mitochondrial binding site for hexokinase in oxidative muscles

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are the main pathway for metabolites across the mitochondrial outer membrane and may serve as binding sites for kinases, including hexokinase. We determined that mitochondria-bound hexokinase activity is significantly reduced in oxidative muscles (heart and soleus) in vdac1(-/-) mice. The activity data were supported by western blot analysis using HK2 specific antibody. To gain more insight into the physiologic mean of the results with the activity data, VDAC deficient mice were subjected to glucose tolerance testing and exercise-induced stress, each of which involves tissue glucose uptake via different mechanisms. vdac1(-/-) mice exhibit impaired glucose tolerance whereas vdac3(-/-) mice have normal glucose tolerance and exercise capacity. Mice lacking both VDAC1 and VDAC3 (vdac1(-/-)/vdac3(-/-)) have reduced exercise capacity together with impaired glucose tolerance. Therefore, we demonstrated a link between VDAC1 mediated mitochondria-bound hexokinase activity and the capacity for glucose clearance.     

VDAC1 serves as a mitochondrial binding site for hexokinase in oxidative muscles

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are the main pathway for metabolites across the mitochondrial outer membrane and may serve as binding sites for kinases, including hexokinase. We determined that mitochondria-bound hexokinase activity is significantly reduced in oxidative muscles (heart and soleus) in vdac1^−/− mice. The activity data were supported by western blot analysis using HK2 specific antibody. To gain more insight into the physiologic mean of the results with the activity data, VDAC deficient mice were subjected to glucose tolerance testing and exercise-induced stress, each of which involves tissue glucose uptake via different mechanisms. vdac1^−/− mice exhibit impaired glucose tolerance whereas vdac3^−/− mice have normal glucose tolerance and exercise capacity. Mice lacking both VDAC1 and VDAC3 (vdac1^−/−/vdac3^−/−) have reduced exercise capacity together with impaired glucose tolerance. Therefore, we demonstrated a link between VDAC1 mediated mitochondria-bound hexokinase activity and the capacity for glucose clearance.     

Allosteric nucleotide-binding site in the mitochondrial NADH:ubiquinone oxidoreductase (respiratory complex I)

The rotenone-insensitive NADH:hexaammineruthenium III (HAR) oxidoreductase reactions catalyzed by bovine heart and Yarrowia lipolytica submitochondrial particles or purified bovine complex I are stimulated by ATP and other purine nucleotides. The soluble fraction of mammalian complex I (FP) and prokaryotic complex I homolog NDH-1 in Paracoccus denitrificans plasma membrane lack stimulation of their activities by ATP. The stimulation appears as a decrease in apparent K(m) values for NADH and HAR. Thus, the "accessory" subunits of eukaryotic complex I bear an allosteric ATP-binding site.     

The nucleotide-binding site of human sphingosine kinase 1

Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly(82) of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an approximately 45-fold higher K(m)((ATP)). We have also shown that Lys(103) is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX(17-21)K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.     

On the role of mi-cK and VDAC in mitochondrial function of heart muscle cells

A two-compartment kinetic model was used to describe reconstituted systems in which mitochondria compete with pyruvate kinase for kinase-generated ADP. The modelling suggests that cytosolic CK deficiency results in a 50% increase in outer mitochondrial membrane permeability.     

DJ-1 null dopaminergic neuronal cells exhibit defects in mitochondrial function and structure: involvement of mitochondrial complex I assembly

DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 null dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 null cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 null cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O(2) consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 null cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease.     

Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild‐type but not PD‐linked mutant parkin supports the biogenesis of a population of mitochondria‐derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin‐ and PINK1‐dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD.     

The non-catalytic nucleotide-binding site of mitochondrial ATPase is localised on the alpha-subunit(s) of factor F1

The incubation of isolated factor F1 with the di-aldehyde derivative of ADP (oxADP) which is formed as a result of ADP treatment by periodate, causes the covalent binding of 0.9--1 molecules of the oxADP with a molecule of the enzyme. This modification of factor F1 is not accompanied by any changes in the ATPase activity of the enzyme. The modification of factor F1 is preceded by the reversible binding of oxADP with the enzyme with a Kd of 80 micro M. ADP partly prevents factor F1 from modification by oxADP. The electrophoresis of modified factor F1 in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that oxADP binds with the alpha-subunit(s) of factor F1. When submitochondrial particles are incubated with [3H]oxADP, the main part of the radioactive label may be discovered in the polypeptide with a molecular weight of some 30 000 which is probably the adenine nucleotides' translocase. The isolation of factor F1 from particles preincubated with [3H]oxADP showed that the membrane-bound factor F1 covalently binds 0.2--0.3 mol of oxADP per mol of enzyme. Here again, all the oxADP is bound with the alpha subunit(s) of factor F1. The modification of membrane-bound factor F1 by oxADP is accompanied by the partial inhibition of the particles' ATPase activity. The results obtained testify to the fact that the non-catalytic site of mitochondrial ATP ase located on the alpha-subunit(s) of factor F1 may participate in the mechanism of ATP hydrolysis by membrane-bound ATPase.     

A new role for ATM: regulating mitochondrial function and mitophagy

The various pathologies in ataxia telangiectasia (A-T) patients including T-cell lymphomagenesis have been attributed to defects in the DNA damage response pathway because ATM, the gene mutated in this disease, is a key mediator of this process. Analysis of Atm-deficient thymocytes in mice reveals that the absence of this gene results in altered mitochondrial homeostasis, a phenomenon that appears to result from abnormal mitophagy engagement. Interestingly, allelic loss of the autophagic gene Becn1 delays tumorigenesis in Atm-null mice presumably by reversing the mitochondrial abnormalities and not by improving the DNA damage response (DDR) pathway. Thus, ATM plays a critical role in modulating mitochondrial homeostasis perhaps by regulating mitophagy.     

Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC oligomerization: release of cytochrome c,AIF and Smac/Diablo

Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H₂O₂ or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.     

Adenine nucleotide-binding sites on mitochondrial F1-ATPase: studies of the inactive complex formed upon binding ADP at a catalytic site.

ADP-induced inhibition of mitochondrial F1-ATPase has been studied. It is shown that in the presence of magnesium and the absence of light, the photoaffinity ADP analog, 2-azido-ADP, induces a reversible inhibition of native F1 that is indistinguishable from that obtained with ADP. Photolysis of the inactive complex results in the predominant labeling of a catalytic-site peptide identified previously (Cross et al., 1987, Proc. Natl. Acad. Sci. USA 84, 5715-5719). Dissociation of the inactive complex formed between F1 and ADP is biphasic with a rapid azide-insensitive phase followed by a slow azide-sensitive phase (k approximately 3 x 10(-3) s-1). It is also shown that incubation of the ADP-inhibited enzyme with EDTA or phosphate does not result in release or migration of ADP from the catalytic site. However, it does convert the complex to a form that reactivates in the presence of 100 microM ATP at a rate too rapid to observe using manual mixing.     

VDAC regulation: role of cytosolic proteins and mitochondrial lipids

It was recently asserted that the voltage-dependent anion channel (VDAC) serves as a global regulator, or governor, of mitochondrial function (Lemasters and Holmuhamedov, Biochim Biophys Acta 1762:181–190, 2006). Indeed, VDAC, positioned on the interface between mitochondria and the cytosol (Colombini, Mol Cell Biochem 256:107–115, 2004), is at the control point of mitochondria life and death. This large channel plays the role of a “switch” that defines in which direction mitochondria will go: to normal respiration or to suppression of mitochondria metabolism that leads to apoptosis and cell death. As the most abundant protein in the mitochondrial outer membrane (MOM), VDAC is known to be responsible for ATP/ADP exchange and for the fluxes of other metabolites across MOM. It controls them by switching between the open and “closed” states that are virtually impermeable to ATP and ADP. This control has dual importance: in maintaining normal mitochondria respiration and in triggering apoptosis when cytochrome c and other apoptogenic factors are released from the intermembrane space into the cytosol. Emerging evidence indicates that VDAC closure promotes apoptotic signals without direct involvement of VDAC in the permeability transition pore or hypothetical Bax-containing cytochrome c permeable pores. VDAC gating has been studied extensively for the last 30 years on reconstituted VDAC channels. In this review we focus exclusively on physiologically relevant regulators of VDAC gating such as endogenous cytosolic proteins and mitochondrial lipids. Closure of VDAC induced by such dissimilar cytosolic proteins as pro-apoptotic tBid and dimeric tubulin is compared to show that the involved mechanisms are rather distinct. While tBid mostly modulates VDAC voltage gating, tubulin blocks the channel with the efficiency of blockage controlled by voltage. We also discuss how characteristic mitochondrial lipids, phospatidylethanolamine and cardiolipin, could regulate VDAC gating. Overall, we demonstrate that VDAC gating is not just an observation made under artificial conditions of channel reconstitution but is a major mechanism of MOM permeability control.     

Supramolecular assembly of VDAC in native mitochondrial outer membranes

The voltage-dependent anion channel (VDAC) is the most abundant protein in the mitochondrial outer membrane (MOM). Due to its localization, VDAC is involved in a wide range of processes, such as passage of ATP out of mitochondria, and particularly plays a central role in apoptosis. Importantly, the assembly of VDAC provides interaction with a wide range of proteins, some implying oligomerization. However, many questions remain as to the VDAC structure, its supramolecular assembly, packing density, and oligomerization in the MOM is unknown. Here we report the so far highest resolution view of VDAC and its native supramolecular assembly. We have studied yeast MOM by high-resolution atomic force microscopy (AFM) in physiological buffer and found VDAC in two distinct types of membrane domains. We found regions where VDAC was packed at high density (approximately 80%), rendering the membrane a voltage-dependent molecular sieve. In other domains, VDAC has a low surface density (approximately 20%) and the pore assembly ranges from single molecules to groups of up to 20. We assume that these groups are mobile in the lipid bilayer and allow association and dissociation with the large assemblies. VDAC has no preferred oligomeric state and no long-range order was observed in densely packed domains. High-resolution topographs show an eye-shaped VDAC with 3.8 nm x 2.7 nm pore dimensions. Based on the observed VDAC structure and the pair correlation function (PCF) analysis of the domain architectures, we propose a simple model that could explain the phase behavior of VDAC, and illustrates the sensitivity of the molecular organization to conditions in the cell, and the possibility for modulation of its assembly. The implication of VDAC in cytochrome c release from the mitochondria during cell apoptosis has made it a target in cancer research.     

Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence.

virB11, one of the 11 genes of the virB operon, is absolutely required for transport of T-DNA from Agrobacterium tumefaciens into plant cells. Previous studies reported that VirB11 is an ATPase with autophosphorylation activity and localizes to the inner membrane even though the protein does not contain the consensus N-terminal export sequence. In this report, we show that VirB11 localizes to the inner membrane even in the absence of other tumor-inducing (Ti) plasmid-encoded proteins. To facilitate the further characterization of VirB11, we purified this protein from the soluble fraction of an Escherichia coli extract by fusing VirB11 to the maltose-binding protein. The maltose-binding protein-VirB11 fusion was able to complement a virB11 deletion mutant of A. tumefaciens for tumor formation and also localized properly to the inner membrane of A. tumefaciens. The 72-kDa protein, purified from E. coli, exhibited no autophosphorylation, ATPase activity, or ATP-binding activity. To study the importance of the Walker nucleotide-binding site present in VirB11, mutations were generated to replace the conserved lysine residue with either alanine or arginine. Expression of the virB11K175A mutant gene resulted in an avirulent phenotype, and expression of the virB11K175R mutant gene gave rise to an attenuated virulence phenotype. Both mutant proteins were present at levels three to four times higher than that of VirB11 in the wild-type strain. The mutant genes did not exhibit a transdominant phenotype on tumor formation in bacteria that were expressing wild-type virB11. The mutant proteins also localized properly to the inner membrane of A. tumefaciens, but the VirB11K175R protein appeared to be unstable after lysis of the cells.