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Membrane Lipid Composition Regulates Tubulin Interaction with Mitochondrial Voltage-dependent Anion Channel*

Open AccessPublished:July 04, 2012DOI:https://doi.org/10.1074/jbc.M112.378778
      Elucidating molecular mechanisms by which lipids regulate protein function within biological membranes is critical for understanding the many cellular processes. Recently, we have found that dimeric αβ-tubulin, a subunit of microtubules, regulates mitochondrial respiration by blocking the voltage-dependent anion channel (VDAC) of mitochondrial outer membrane. Here, we show that the mechanism of VDAC blockage by tubulin involves tubulin interaction with the membrane as a critical step. The on-rate of the blockage varies up to 100-fold depending on the particular lipid composition used for bilayer formation in reconstitution experiments and increases with the increasing content of dioleoylphosphatidylethanolamine (DOPE) in dioleoylphosphatidylcholine (DOPC) bilayers. At physiologically low salt concentrations, the on-rate is decreased by the charged lipid. The off-rate of VDAC blockage by tubulin does not depend on the lipid composition. Using confocal fluorescence microscopy, we compared tubulin binding to the membranes of giant unilamellar vesicles (GUVs) made from DOPC and DOPC/DOPE mixtures. We found that detectable binding of the fluorescently labeled dimeric tubulin to GUV membranes requires the presence of DOPE. We propose that prior to the characteristic blockage of VDAC, tubulin first binds to the membrane in a lipid-dependent manner. We thus reveal a new potent regulatory role of the mitochondrial lipids in control of the mitochondrial outer membrane permeability and hence mitochondrial respiration through tuning VDAC sensitivity to blockage by tubulin. More generally, our findings give an example of the lipid-controlled protein-protein interaction where the choice of lipid species is able to change the equilibrium binding constant by orders of magnitude.

      Introduction

      The ability of lipids to regulate proteins arises from both specific chemical features of lipid molecules and mechanical and structural properties of the lipid bilayer (
      • Marsh D.
      Protein modulation of lipids, and vice versa, in membranes.
      ,
      • McIntosh T.J.
      • Simon S.A.
      Roles of bilayer material properties in function and distribution of membrane proteins.
      ,
      • Smith A.W.
      Lipid-protein interactions in biological membranes. A dynamic perspective.
      ,
      • Lee A.G.
      Lipid-protein interactions in biological membranes. A structural perspective.
      ,
      • Cullis P.R.
      • de Kruijff B.
      Lipid polymorphism and the functional roles of lipids in biological membranes.
      ,
      • Valiyaveetil F.I.
      • Zhou Y.
      • MacKinnon R.
      Lipids in the structure, folding, and function of the KcsA K+ channel.
      ,
      • Vitrac H.
      • Bogdanov M.
      • Heacock P.
      • Dowhan W.
      Lipids and topological rules of membrane protein assembly. Balance between long and short range lipid-protein interactions.
      ). Structural, compositional, and elastic parameters of lipid membranes are known to have a strong influence on the function of membrane proteins, such as ion channels, as well as on the interaction of water-soluble proteins with membranes. Hydrophobic mismatch between the acyl chain region of the membrane and the embedded proteins directly influences ion channel behavior (
      • Valiyaveetil F.I.
      • Zhou Y.
      • MacKinnon R.
      Lipids in the structure, folding, and function of the KcsA K+ channel.
      ,
      • Lundbaek J.A.
      • Collingwood S.A.
      • Ingólfsson H.I.
      • Kapoor R.
      • Andersen O.S.
      Lipid bilayer regulation of membrane protein function. Gramicidin channels as molecular force probes.
      ,
      • Schmidt D.
      • Jiang Q.X.
      • MacKinnon R.
      Phospholipids and the origin of cationic gating charges in voltage sensors.
      ,
      • Xu Y.
      • Ramu Y.
      • Lu Z.
      Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels.
      ,
      • Yoshimura K.
      • Sokabe M.
      Mechanosensitivity of ion channels based on protein-lipid interactions.
      ). There is also clear evidence of the strong response of ion channels to the elastic stress within a lipid bilayer or the lipid packing stress (
      • Bezrukov S.M.
      Functional consequences of lipid packing stress.
      ,
      • van den Brink-van der Laan E.
      • Killian J.A.
      • de Kruijff B.
      Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile.
      ,
      • Sobko A.A.
      • Kotova E.A.
      • Antonenko Y.N.
      • Zakharov S.D.
      • Cramer W.A.
      Effect of lipids with different spontaneous curvature on the channel activity of colicin E1. Evidence in favor of a toroidal pore.
      ). Phosphatidylcholine (PC)
      The abbreviations used are: PC
      phosphatidylcholine
      PE
      phosphatidylethanolamine
      MOM
      mitochondrial outer membrane
      VDAC
      voltage-dependent anion channel
      DOPE
      dioleoylphosphatidylethanolamine
      DOPC
      dioleoylphosphatidylcholine
      CTT
      C-terminal tail
      PLE
      polar lipid extract
      GUV
      giant unilamellar vesicle
      CL
      cardiolipin
      DPhPC
      diphytanoyl-phosphatidylcholine
      DOTAP
      dioleoyl-trimethylammonium-propane
      DPhPS
      diphytanoyl-phosphatidylserine.
      and phosphatidylethanolamine (PE) lipids commonly found in cell membranes are the main components of the mitochondrial outer membrane (MOM) (
      • Daum G.
      Lipids of mitochondria.
      ,
      • Ardail D.
      • Privat J.P.
      • Egret-Charlier M.
      • Levrat C.
      • Lerme F.
      • Louisot P.
      Mitochondrial contact sites. Lipid composition and dynamics.
      ,
      • de Kroon A.I.
      • Dolis D.
      • Mayer A.
      • Lill R.
      • de Kruijff B.
      Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane?.
      ). Most of PC lipids are lamellar lipids that form “flat” bilayers, whereas nonlamellar PE lipids tend to form highly curved nonbilayer phases. When forced into a flat bilayer structure, PE lipids produce a significant stress in the hydrocarbon area of the membrane (
      • van den Brink-van der Laan E.
      • Killian J.A.
      • de Kruijff B.
      Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile.
      ,
      • Gruner S.M.
      Intrinsic curvature hypothesis for biomembrane lipid-composition. A role for nonbilayer lipids.
      ,
      • Bezrukov S.M.
      • Rand R.P.
      • Vodyanoy I.
      • Parsegian V.A.
      Lipid packing stress and polypeptide aggregation. Alamethicin channel probed by proton titration of lipid charge.
      ,
      • Cantor R.S.
      Lipid composition and the lateral pressure profile in membranes.
      ). For example, inclusion of PE into a PC bilayer increases the lateral pressure in the hydrophobic core of the bilayer with a compensating decrease of the pressure in the headgroup region. This leads to a redistribution of the lateral pressure in the membrane. Here, we use various PE/PC compositions to manipulate lipid packing stress and to study how this affects the interaction of two proteins: a membrane protein, voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane, and a water-soluble protein, dimeric tubulin. We took advantage of our recent finding that dimeric tubulin reversibly and with high efficiency blocks VDAC reconstituted into planar lipid membranes (
      • Rostovtseva T.K.
      • Bezrukov S.M.
      VDAC regulation. Role of cytosolic proteins and mitochondrial lipids.
      ,
      • Rostovtseva T.K.
      • Sheldon K.L.
      • Hassanzadeh E.
      • Monge C.
      • Saks V.
      • Bezrukov S.M.
      • Sackett D.L.
      Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration.
      ,
      • Rostovtseva T.K.
      • Bezrukov S.M.
      VDAC inhibition by tubulin and its physiological implications.
      ). Both proteins, as shown previously, are affected by lipid membrane composition.
      The ability of the water-soluble αβ-heterodimer of tubulin to bind to cell membranes with affinity of nm−1 was reported some 30 years ago. Even earlier, it had been shown that tubulin could also bind to liposome membranes, and surprisingly for a water-soluble protein, this tubulin-membrane interaction was proposed to have a hydrophobic component (
      • Klausner R.D.
      • Kumar N.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 1. Association with vesicles at the phase transition.
      ,
      • Kumar N.
      • Klausner R.D.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 2. Physical changes of the protein.
      ,
      • Caron J.M.
      • Berlin R.D.
      Interaction of microtubule proteins with phospholipid vesicles.
      ,
      • Caron J.M.
      • Berlin R.D.
      Reversible adsorption of microtubule protein to phospholipid vesicles.
      ). Importantly, tubulin, a cytoplasmic protein and the basic structural unit of microtubules, was found to be associated with various cell membranes, including mitochondrial membranes (
      • Klausner R.D.
      • Kumar N.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 1. Association with vesicles at the phase transition.
      ,
      • Wolff J.
      Plasma membrane tubulin.
      ,
      • Bernier-Valentin F.
      • Aunis D.
      • Rousset B.
      Evidence for tubulin-binding sites on cellular membranes. Plasma membranes, mitochondrial membranes, and secretory granule membranes.
      ), and it was even suggested as their inherent component (
      • Carré M.
      • André N.
      • Carles G.
      • Borghi H.
      • Brichese L.
      • Briand C.
      • Braguer D.
      Tubulin is an inherent component of mitochondrial membranes that interacts with the voltage-dependent anion channel.
      ). A variety of reported interactions between tubulin and liposomes demonstrated a potential for both integral and surface attachments of tubulin to phospholipid bilayers, although it remains unclear how tubulin becomes an integral component of cell membranes. Tubulin was shown to insert into the bilayers of saturated phosphatidylcholine vesicles and form stable vesicle-tubulin complexes without any requirements of detergent or sonication (
      • Klausner R.D.
      • Kumar N.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 1. Association with vesicles at the phase transition.
      ,
      • Kumar N.
      • Klausner R.D.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 2. Physical changes of the protein.
      ,
      • Caron J.M.
      • Berlin R.D.
      Interaction of microtubule proteins with phospholipid vesicles.
      ). The insertion process is accompanied by structural perturbations of both tubulin and lipid bilayer and involves hydrophobic interactions (
      • Klausner R.D.
      • Kumar N.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 1. Association with vesicles at the phase transition.
      ,
      • Kumar N.
      • Klausner R.D.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 2. Physical changes of the protein.
      ). It was also demonstrated that tubulin could reversibly adsorb to PC or PS membranes above the lipid phase transition (
      • Caron J.M.
      • Berlin R.D.
      Dynamic interactions between microtubules and artificial membranes.
      ). There are strong indications that tubulin-lipid interactions are highly lipid-specific. Although tubulin binding to phospholipid membranes has been extensively studied, the physiological role of this interaction remains poorly understood (
      • Wolff J.
      Plasma membrane tubulin.
      ).
      Voltage gating of VDAC reconstituted into planar membranes was shown to depend on the presence of nonlamellar lipids (
      • Rostovtseva T.K.
      • Bezrukov S.M.
      VDAC regulation. Role of cytosolic proteins and mitochondrial lipids.
      ,
      • Rostovtseva T.K.
      • Kazemi N.
      • Weinrich M.
      • Bezrukov S.M.
      Voltage gating of VDAC is regulated by nonlamellar lipids of mitochondrial membranes.
      ). Previously, we have found that nonlamellar lipids, characteristic for mitochondrial membrane, PE, and cardiolipin (CL), change VDAC conformational equilibrium to promote the low conducting “closed” states at negative potentials, suggesting a coupling between the mechanical pressure in the hydrocarbon region of the lipid bilayer and the voltage-induced conformational transitions of VDAC.
      VDAC, the major channel in MOM, not only serves as a principal pathway for ATP, ADP, and other mitochondrial respiratory substrates across MOM but also controls these fluxes, and thus it plays the role of a global regulator of mitochondrial functions and cell metabolism (
      • Lemasters J.J.
      • Holmuhamedov E.
      Voltage-dependent anion channel (VDAC) as mitochondrial governator. Thinking outside the box.
      ,
      • Rostovtseva T.K.
      • Tan W.
      • Colombini M.
      On the role of VDAC in apoptosis. Fact and fiction.
      ,
      • Colombini M.
      VDAC. The channel at the interface between mitochondria and the cytosol.
      ,
      • Shoshan-Barmatz V.
      • De Pinto V.
      • Zweckstetter M.
      • Raviv Z.
      • Keinan N.
      • Arbel N.
      VDAC, a multifunctional mitochondrial protein regulating cell life and death.
      ). Recently, we have found that in the presence of tubulin, the conductance of VDAC reconstituted into a planar lipid membrane fluctuates between the open and tubulin-blocked state (
      • Rostovtseva T.K.
      • Sheldon K.L.
      • Hassanzadeh E.
      • Monge C.
      • Saks V.
      • Bezrukov S.M.
      • Sackett D.L.
      Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration.
      ). Although the tubulin-blocked state is still conductive for small ions (about 40% of the open state conductance in 1 m KCl), it has reduced dimensions compared with the open state, reversed ionic selectivity, and most importantly is virtually impermeable for ATP (
      • Gurnev P.A.
      • Rostovtseva T.K.
      • Bezrukov S.M.
      Tubulin-blocked state of VDAC studied by polymer and ATP partitioning.
      ). These data strongly suggest that not only ATP, but ADP and other mitochondrial respiratory substrates, most of which are negatively charged and are close to or larger than ATP by their molecular weight, cannot permeate through the tubulin-blocked state due to the steric restrictions and the electrostatic barrier. It was concluded that by blocking VDAC permeability, tubulin may selectively regulate fluxes of metabolites across MOM and therefore control mitochondrial respiration. Indeed, experiments with isolated mitochondria (
      • Rostovtseva T.K.
      • Sheldon K.L.
      • Hassanzadeh E.
      • Monge C.
      • Saks V.
      • Bezrukov S.M.
      • Sackett D.L.
      Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration.
      ,
      • Monge C.
      • Beraud N.
      • Kuznetsov A.V.
      • Rostovtseva T.
      • Sackett D.
      • Schlattner U.
      • Vendelin M.
      • Saks V.A.
      Regulation of respiration in brain mitochondria and synaptosomes. Restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase.
      ) and with intact cells (
      • Maldonado E.N.
      • Patnaik J.
      • Mullins M.R.
      • Lemasters J.J.
      Free tubulin modulates mitochondrial membrane potential in cancer cells.
      ) supported this conjecture.
      Earlier, we proposed a model of VDAC-tubulin interaction where the negatively charged C-terminal tail (CTT) of tubulin permeates into the channel lumen, interacting with VDAC and reversibly blocking channel conductance in a highly voltage-dependent manner (
      • Rostovtseva T.K.
      • Sheldon K.L.
      • Hassanzadeh E.
      • Monge C.
      • Saks V.
      • Bezrukov S.M.
      • Sackett D.L.
      Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration.
      ,
      • Rostovtseva T.K.
      • Bezrukov S.M.
      VDAC inhibition by tubulin and its physiological implications.
      ). Here, we report that the mechanism of the VDAC-tubulin interaction is more complex than was initially thought and, what may seem to be unexpected, strongly depends on the specific composition of the lipid membrane. We find that the on-rate of VDAC blockage by tubulin varies up to 100-fold between DOPC and DOPE membranes, increasing with the PE content. VDAC-tubulin interaction depends on both hydrophobic and polar parts of the lipid molecule. Our data imply that the previously shown ability of tubulin to bind to lipid membranes in a saturable, reversible, and specific manner (
      • Wolff J.
      Plasma membrane tubulin.
      ,
      • Bernier-Valentin F.
      • Aunis D.
      • Rousset B.
      Evidence for tubulin-binding sites on cellular membranes. Plasma membranes, mitochondrial membranes, and secretory granule membranes.
      ) could greatly impact VDAC blockage by tubulin. At a physiologically low salt concentration of 100 mm KCl, we found that the charge of lipid headgroups significantly affects the on-rate of the blockage too. Finally, using confocal microscopy of giant unilamellar vesicles (GUVs) in the presence of fluorescently labeled dimeric tubulin, we demonstrated that measurable adsorption of tubulin requires the presence of PE. Thus, our findings suggest a new regulatory role of the mitochondrial lipids in control of MOM permeability and mitochondrial respiration through the lipid-mediated tuning of VDAC sensitivity to tubulin.

      DISCUSSION

      The model of VDAC blockage by tubulin proposed earlier (
      • Rostovtseva T.K.
      • Sheldon K.L.
      • Hassanzadeh E.
      • Monge C.
      • Saks V.
      • Bezrukov S.M.
      • Sackett D.L.
      Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration.
      ,
      • Rostovtseva T.K.
      • Hassanzadeh E.
      • Sackett D.L.
      • Bezrukov S.M.
      Tubulin regulates VDAC channel.
      ) took into account only direct interaction between the two proteins and did not imply any involvement of lipids. The remarkable sensitivity of the blockage to the phosphorylation state of VDAC reported later (
      • Sheldon K.L.
      • Maldonado E.N.
      • Lemasters J.J.
      • Rostovtseva T.K.
      • Bezrukov S.M.
      Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin.
      ) gave additional support for the model. In this respect, the strong effect of the membrane lipid composition reported here was quite unexpected. This finding suggested that the original model was missing an important step of tubulin adsorption to the membrane, and this step of the reaction is the main cause for the observed sensitivity of the blockage to the lipid composition. Such a conclusion is supported by the preferential adsorption of the fluorescently labeled tubulin-488 to the PE-containing GUV membranes as compared with the GUV membranes made of pure DOPC (Fig. 5). The apparent paradox of tubulin, a soluble protein, interacting with neutral lipid membranes has been recognized much earlier (
      • Klausner R.D.
      • Kumar N.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 1. Association with vesicles at the phase transition.
      ,
      • Kumar N.
      • Klausner R.D.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 2. Physical changes of the protein.
      ,
      • Caron J.M.
      • Berlin R.D.
      Interaction of microtubule proteins with phospholipid vesicles.
      ). It was proposed that this interaction has a hydrophobic nature, in which both tubulin and lipid undergo significant conformational changes upon binding (
      • Klausner R.D.
      • Kumar N.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 1. Association with vesicles at the phase transition.
      ,
      • Kumar N.
      • Klausner R.D.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 2. Physical changes of the protein.
      ,
      • Caron J.M.
      • Berlin R.D.
      Interaction of microtubule proteins with phospholipid vesicles.
      ). Importantly, it was shown that the membrane-bound tubulin is not functionally “denatured” because it was still able to bind colchicine and microtubule-associated proteins with an unaltered rate (
      • Klausner R.D.
      • Kumar N.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 1. Association with vesicles at the phase transition.
      ).
      In this study, we find that another functionally important property of tubulin is not compromised in the membrane-bound state, namely its ability to regulate VDAC. In what seems to be most relevant to this study, it was found that CTTs of liposome-associated tubulin were accessible to proteolysis (
      • Hargreaves A.J.
      • McLean W.G.
      The characterization of phospholipids associated with microtubules, purified tubulin and microtubule associated proteins in vitro.
      ), which indicates that tubulin CTTs are not directly involved in the interaction with membranes and, in our case, could be available for blocking VDAC pore following a proposed earlier model where tubulin CTTs are required for VDAC blockage (
      • Rostovtseva T.K.
      • Sheldon K.L.
      • Hassanzadeh E.
      • Monge C.
      • Saks V.
      • Bezrukov S.M.
      • Sackett D.L.
      Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration.
      ).
      Although the mechanism of tubulin interaction with membranes is complex and yet not understood (
      • Wolff J.
      Plasma membrane tubulin.
      ), one of the possibilities is that lipid packing stress could influence tubulin-membrane association. From x-ray diffraction, it was inferred that the repulsive forces between DOPE headgroups are significantly smaller than those between DOPC headgroups (
      • Keller S.L.
      • Bezrukov S.M.
      • Gruner S.M.
      • Tate M.W.
      • Vodyanoy I.
      • Parsegian V.A.
      Probability of alamethicin conductance states varies with nonlamellar tendency of bilayer phospholipids.
      ). We hypothesize that the decrease in the repulsive forces between DOPE headgroups provides for a more flexible arrangement of the groups on the membrane surface, thus making the hydrophobic region of the membrane more accessible for its interaction with tubulin. This may explain the differences in tubulin binding to DOPC and DOPE membranes (Fig. 5) and the corresponding differences in the VDAC blockage on-rates (Fig. 2, A and C). Preferential association of tubulin with PE-containing membranes results in the increased effective concentration of tubulin on the surface of these membranes, which is seen as the increased on-rate of the blockage.
      The hydrophobic component of tubulin interaction with the membrane might involve a partial insertion of tubulin hydrophobic domains into the membrane (
      • Kumar N.
      • Klausner R.D.
      • Weinstein J.N.
      • Blumenthal R.
      • Flavin M.
      Interaction of tubulin with phospholipid-vesicles. 2. Physical changes of the protein.
      ). Thus, the effect of lipid on the VDAC blockage by tubulin could be described by a tentative model shown in Fig. 6, where the first step is a saturable lipid-dependent tubulin binding to the membrane. Some lipids such as DPhPC and DOPE, are “tubulin-sticky” and thus increase its concentration around VDAC, whereas other lipids, such as DOPC or PLE, are not. The second step is interaction of tubulin with the cytosolic loops of VDAC, which depends on their phosphorylation state (
      • Sheldon K.L.
      • Maldonado E.N.
      • Lemasters J.J.
      • Rostovtseva T.K.
      • Bezrukov S.M.
      Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin.
      ). The final step is a partial block of the VDAC pore by tubulin CTT. The on-rate of this final step of blockage should depend on the effective concentration of tubulin tails at the membrane surface in a close proximity to the VDAC entrance and consequently on tubulin binding to the membrane surface and to the cytosolic loops of VDAC. In principle, both step 1 and step 2 can be voltage-dependent. We do not show this dependence in Fig. 6 because, at the moment, we do not have any strong support for such a conjecture. Regarding step 3, it is definitely voltage-dependent (Fig. 2A). Although the on-rate voltage dependence can be explained by hypothetical voltage dependences of step 1 and/or step 2, the off-rate shows profound voltage sensitivity (Fig. 2B). It is important that the off-rate is defined by the applied voltage and interactions between CTT and the VDAC pore and is independent of the VDAC phosphorylation state (
      • Sheldon K.L.
      • Maldonado E.N.
      • Lemasters J.J.
      • Rostovtseva T.K.
      • Bezrukov S.M.
      Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin.
      ), tubulin concentration in the bulk (
      • Rostovtseva T.K.
      • Sheldon K.L.
      • Hassanzadeh E.
      • Monge C.
      • Saks V.
      • Bezrukov S.M.
      • Sackett D.L.
      Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration.
      ,
      • Rostovtseva T.K.
      • Bezrukov S.M.
      VDAC inhibition by tubulin and its physiological implications.
      ), and tubulin interaction with the membrane (Fig. 2B). Further refinement of the model, as well as quantitative analysis of the kinetics involved in the first two steps, will require specially designed relaxation experiments.
      Figure thumbnail gr6
      FIGURE 6A tentative model of the multistep VDAC blockage by tubulin. Step 1 is lipid-sensitive tubulin binding to the membrane. Step 2 is the phosphorylation-sensitive interaction of tubulin with the VDAC cytosolic loops (
      • Sheldon K.L.
      • Maldonado E.N.
      • Lemasters J.J.
      • Rostovtseva T.K.
      • Bezrukov S.M.
      Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin.
      ). Step 3 is a voltage-dependent partial block of VDAC pore by tubulin CTT (
      • Rostovtseva T.K.
      • Sheldon K.L.
      • Hassanzadeh E.
      • Monge C.
      • Saks V.
      • Bezrukov S.M.
      • Sackett D.L.
      Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration.
      ). Both the off-rate and on-rate of this final step of the blockage are strongly voltage-sensitive, with the on-rate increasing with the increase of the effective concentration of tubulin tails at the membrane surface in a close proximity to the VDAC entrance.
      The distribution of times characterizing the on-rate of VDAC blockage by tubulin is well described by a single exponential fitting (Fig. 1B). There is no indication of the presence of the second time constant to account for the tubulin-membrane binding kinetics. Therefore, the steps of tubulin binding to the membrane and/or to the VDAC cytosolic loops should be either much slower or much faster than the characteristic VDAC open time found to be in a range of 0.1–10 s (Fig. 1B). Slow kinetics of tubulin binding to the membrane, in the range of more than 100 s, seems to be the most likely scenario, because usually about 5–10 min after tubulin addition are required for blockage events to reach a steady-state frequency. Indeed, even slower kinetics, up to 40 min, were reported for the 125I-labeled tubulin binding to the liposomes formed from plasma membrane fractions (
      • Bernier-Valentin F.
      • Aunis D.
      • Rousset B.
      Evidence for tubulin-binding sites on cellular membranes. Plasma membranes, mitochondrial membranes, and secretory granule membranes.
      ). Experiments presented in Fig. 3 were carried out following a “quasi-stationary” protocol where τon was measured ∼5 min after each tubulin addition to the same membrane. Five minutes appeared to be sufficient for tubulin binding to reach equilibrium when membranes were made from a “sticky” DPhPC lipid, because a steady saturation level was reached at tubulin concentrations higher than 50 nm (Fig. 3). When DOPC included 75% of the total lipid in the DOPC/DOPE mixture, the equilibration was much slower. The absence of a clear saturation in Fig. 3 (see inset) for the DOPC/DOPE mixture could be partially related to this slow kinetics that required times exceeding those experimentally attainable in single channel recording.
      In the PLE membranes, with the lipid mixture that closely resembles the MOM lipid content, the on-rate of VDAC blockage by tubulin was even less than in pure DOPC membranes (Fig. 2A). This observation helps to resolve an apparent discrepancy between our in vitro data, where tubulin could block VDAC conductance at nanomolar concentrations, and the fact that there is up to 20 μm free tubulin in some cells (
      • Gard D.L.
      • Kirschner M.W.
      Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs.
      ). Depending on the PC/PE ratio in the membrane, the on-rate constant of the blockage could be changed by orders of magnitude, and at potentials across MOM close to zero, micromoles of tubulin would be required to block VDAC in vivo. Because the off-rates do not depend on the lipid composition, the changes in the equilibrium constant Keq are primarily defined by the changes in the on-rate; Keq increases 100 times when DOPC is replaced by DOPE (Fig. 2C). Previously, we have shown that Keq of VDAC blockage by tubulin spans 6 orders of magnitude from nm−1 to mm−1 depending on the applied voltage and the degree of VDAC phosphorylation (
      • Sheldon K.L.
      • Maldonado E.N.
      • Lemasters J.J.
      • Rostovtseva T.K.
      • Bezrukov S.M.
      Phosphorylation of voltage-dependent anion channel by serine/threonine kinases governs its interaction with tubulin.
      ). Here, we find that lipid composition could also significantly shift the equilibrium constant of the blockage. Considering a substantial lipid homeostasis in mitochondria during fusion and fission (
      • Furt F.
      • Moreau P.
      Importance of lipid metabolism for intracellular and mitochondrial membrane fusion/fission processes.
      ), under apoptotic stress (
      • Crimi M.
      • Esposti M.D.
      Apoptosis-induced changes in mitochondrial lipids.
      ) and mitochondrial lipid oxidation (
      • Daum G.
      Lipids of mitochondria.
      ,
      • Pamplona R.
      Membrane phospholipids, lipoxidative damage, and molecular integrity. A causal role in aging and longevity.
      ,
      • Paradies G.
      • Petrosillo G.
      • Paradies V.
      • Ruggiero F.M.
      Mitochondrial dysfunction in brain aging. Role of oxidative stress and cardiolipin.
      ,
      • Kagan V.E.
      • Borisenko G.G.
      • Tyurina Y.Y.
      • Tyurin V.A.
      • Jiang J.
      • Potapovich A.I.
      • Kini V.
      • Amoscato A.A.
      • Fujii Y.
      Oxidative lipidomics of apoptosis. Redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine.
      ), lipids could be distinct regulators of the VDAC-tubulin interaction and therefore MOM permeability in vivo.
      According to the proposed lipid-dependent step of VDAC blockage by tubulin (Fig. 6), the modified tubulin-membrane association would result in the different on-rates in the membranes formed with different solvents. The presence of residual amounts of hexadecane in the hydrophobic part of the DPhPC bilayer could enhance VDAC blockage by tubulin by enforcing the hydrophobic component of the tubulin-membrane interaction.
      Depending on the particular conditions, such as salt concentration and lipid type, one of the components of the tubulin-membrane interaction could suppress or synergistically reinforce another. For instance, electrostatic repulsion between acidic tubulin and negatively charged PS in DPhPS/DPhPC membranes at low salt concentrations is expected to suppress the hydrophobic interactions; accordingly, the presence of PS results in a 100 times reduced kon in comparison with neutral DPhPC membranes (Fig. 4A). In the DOTAP-containing membranes, the positively charged lipid could compete for the negatively charged CTT thus making them less available for VDAC blockage. At the same time, hydrophobic forces could compensate and overcome electrostatic repulsion between tubulin and DOTAP at high salt concentrations (Fig. 4B). It has to be noted here that nonlamellar tendency of charged lipids is a function of salt concentration (
      • Bezrukov S.M.
      • Rand R.P.
      • Vodyanoy I.
      • Parsegian V.A.
      Lipid packing stress and polypeptide aggregation. Alamethicin channel probed by proton titration of lipid charge.
      ), which further complicates the comparison of the results obtained under different salt conditions. Indeed, there is a possibility that low salts change the lipid packing stress of the DPhPS/DPhPC membranes, which synergistically with electrostatic repulsion reduces kon in comparison with the DPhPC membranes.
      In any case, our results demonstrate that the lipid-dependent tubulin binding to the membrane greatly impacts VDAC blockage by tubulin. It seems reasonable to suggest that hydrophobic interactions between the tubulin membrane-embedded domain and the nonpolar part of the membrane represent an essential component of the multistep VDAC blockage by tubulin. These interactions are sensitive to the bilayer mechanical parameters such as the hydrophobic thickness and lipid packing stress that depend on both the polar headgroup and hydrophobic acyl chains of the lipid. At physiologically low salt concentrations, tubulin-membrane association and consequently VDAC-tubulin interaction may also be modified by Coulomb forces between the lipid headgroup and tubulin charges.
      To conclude, our findings suggest a new regulatory role of mitochondrial lipids in control of MOM permeability, and hence mitochondrial respiration, by tuning of VDAC sensitivity to blockage by tubulin. More generally, they give a clear example of lipid-assisted protein-protein interaction which, quantified by the equilibrium binding constant, can be varied by orders of magnitude through the choice of lipid species.

      Acknowledgments

      We thank Kamran Melikov and Leonid Chernomordik for their assistance with confocal microscopy.

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