Cardiolipin-containing Liposomes Research Articles

Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c.

A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as measured by calcein leakage from liposomes, was observed only in the case of four substitutions in the red Ω-loop (M4 mutant). According to resonance and surface-enhanced Raman spectroscopy, the mutations caused significant changes in the heme of oxidized cytochrome c molecules resulting in an increased probability of the plane heme conformation and the enhancement of the rigidity of the protein surrounding the heme. The binding of wild-type and mutant forms of oxidized cytochrome c to cardiolipin-containing liposomes caused the disordering of the acyl lipid chains that was more pronounced for the M4 mutant. Our findings indicate that the Ω-loop is important for the pore formation in cardiolipin-containing membranes.

The enzyme activity of mitochondrial trifunctional protein is not altered by lysine acetylation or lysine succinylation.

Mitochondrial trifunctional protein (TFP) is a membrane-associated heterotetramer that catalyzes three of the four reactions needed to chain-shorten long-chain fatty acids inside the mitochondria. TFP is known to be heavily modified by acetyllysine and succinyllysine post-translational modifications (PTMs), many of which are targeted for reversal by the mitochondrial sirtuin deacylases SIRT3 and SIRT5. However, the functional significance of these PTMs is not clear, with some reports showing TFP gain-of-function and some showing loss-of-function upon increased acylation. Here, we mapped the known SIRT3/SIRT5-targeted lysine residues onto the recently solved TFP crystal structure which revealed that many of the target sites are involved in substrate channeling within the TFPα subunit. To test the effects of acylation on substate channeling through TFPα, we enzymatically synthesized the physiological long-chain substrate (2E)-hexadecenoyl-CoA. Assaying TFP in SIRT3 and SIRT5 knockout mouse liver and heart mitochondria with (2E)-hexadecenoyl-CoA revealed no change in enzyme activity. Finally, we investigated the effects of lysine acylation on TFP membrane binding in vitro. Acylation did not alter recombinant TFP binding to cardiolipin-containing liposomes. However, the presence of liposomes strongly abrogated the acylation reaction between succinyl-CoA and TFP lysine residues. Thus, TFP in the membrane-bound state may be protected against lysine acylation.

Study of Interaction of Fluorescent Cytochrome C with Liposomes, Mitochondria, and Mitoplasts by Fluorescence Correlation Spectroscopy

Here, we studied the interaction of Cys-substituted (G56C) cytochrome c labeled with sulfocyanin-3 fluorescent dye (fCyt) with artificial and natural lipid membranes by using fluorescence correlation spectroscopy (FCS). Compared to mitochondria, mitoplasts were shown to have more fCyt binding sites with a lower affinity for this protein. The fCyt affinity for cardiolipin-containing liposomes depended on the content of cardiolipin in liposomes and decreased upon raising the ionic strength of the solution. A high value of the constant of the fCyt binding with mitochondria could be explained by the presence of specific binding sites for this protein on the mitochondrial outer membrane surface. This explanation is confirmed by observation of more efficient displacement of fluorescent cytochrome c by the unlabeled mutant variant K8T than by the WT protein, whereas in mitoplasts and liposomes the WT protein displaced fCyt more efficiently than K8T.

PH-Induced Switch between Different Modes of Cytochrome c Binding to Cardiolipin-Containing Liposomes.

Fluorescence, visible circular dichroism (CD), absorption, and resonance Raman spectroscopy techniques were combined to explore structural changes of ferricytochrome c upon its binding to cardiolipin-containing liposomes (20% 1,1′,1,2′-tetraoleyolcardiolipin and 1,2-deoleyol-sn-glycero-3-phosphocholine) at acidic pH (6.5). According to the earlier work of Kawai [J. Biol. Chem.2005, 280, 34709–347171],16012169cytochrome c binding at this pH is governed by interactions between the phosphate head groups of cardiolipin and amino acid side chains of the so-called L-site, which contains the charged residues K22, K25, K27, and potentially H26 and H33. We found that L-site binding causes a conformational transition that involves a change of the protein’s ligation and spin state. In this paper, we report spectroscopic responses to an increasing number of cardiolipin-containing liposomes at pH 6.5 in the absence and presence of NaCl. The latter was found to mostly inhibit protein binding already with 50 mM concentration. The inhibition effect can be quantitatively reproduced by applying the electrostatic theory of Heimburg [Biophys. J.1995, 68, 536–546].7696507 A comparison with corresponding spectroscopic response data obtained at pH 7.4 reveals major differences in that the latter indicates hydrophobic binding, followed by an electrostatically driven conformational change. Visible CD data suggest that structural changes in the heme pocket of liposome-bound ferricytochrome c resemble to some extent those in the denatured protein in urea at neutral and acidic pH. The measured noncoincidence between absorption and CD Soret band of cytochrome c in the presence of a large access of cardiolipin is caused by the electric field at the membrane surface. The very fact that its contribution to the internal electric field in the heme pocket is detectable by spectroscopic means suggests some penetration of the protein into membrane surface.

Cytochrome c-mediated peroxidative permeabilization of cardiolipin-containing liposomes is prevented by minocycline

Cytochrome c-mediated peroxidative permeabilization of cardiolipin-containing liposomes is prevented by minocycline

Relating the multi-functionality of cytochrome c to membrane binding and structural conversion.

Cytochrome c is known as an electron-carrying protein in the respiratory chain of mitochondria. Over the last 20years, however, alternative functions of this very versatile protein have become the focus of research interests. Upon binding to anionic lipids such as cardiolipin, the protein acquires peroxidase activity. Multiple lines of evidence suggest that this requires a conformational change of the protein which involves partial unfolding of its tertiary structure. This review summarizes the current state of knowledge of how cytochrome c interacts with cardiolipin-containing surfaces and how this affects its structure and function. In this context, we delineate partially conflicting results regarding the affinity of cytochrome c binding to cardiolipin-containing liposomes of different size and its influence on the structure of the protein and the morphology of the membrane.

Oxidation State and PH Dependence of Cytochrome C Binding to Cardiolipin-Containing Liposomes

A combination of UV/visible absorption, circular dichroism and fluorescence spectroscopies was used to probe the binding of cytochrome c to cardiolipin (CL) containing liposomes and concomitant conformational changes as a function of cardiolipin concentration at different pH values. Our group has recently developed a model that describes oxidized cytochrome c binding to CL-containing liposomes as a two-step process where native-like liposome bound conformers convert into more unfolded conformations. We have provided evidence that the M80 ligand in the more unfolded state has been replaced by either a lysine or histidine as the axial ligand. A slightly modified binding model was used to describe the binding of reduced cytochrome c, to account for the redox properties of the heme in the absence and presence of oxygen. In our current study, we explored how lowering the pH to values between 6.0 and 7.0 affects binding and conformation of cytochrome c in both oxidation states. It has recently been proposed that such a protocol would facilitate the so-called L-site binding, which involves the electrostatic interaction of amino acid residues Lys22, −25, −27 and His26 and −33 with the acidic phospholipids on the liposome surface. Our results for binding at pH 6.5 indicate a conversion from a low spin to a hexacoordinated high-spin state at moderate to high cardiolipin concentrations, which leads to a significant blue shift of the Soret band. This very much resembles the behavior of denatured cytochrome c where lowering the pH leads to the protonation of a distal histidine ligand and its replacement by a water ligand. Thus, our observations strongly suggest that histidine rather than lysine is the sixth ligand in the misligated state of cytochrome c on CL-containing liposomes.

Ferrocyanide‐Mediated Photoreduction of Ferricytochrome C Utilized to Selectively Probe Non‐native Conformations Induced by Binding to Cardiolipin‐Containing Liposomes

Ferricytochrome c binding to cardiolipin-containing liposomes produces a heterogeneous distribution of conformations comprising native-like and non-native misfolded proteins. We utilized the photoreduction of native ferricytochrome c in the presence of potassium ferrocyanide and resonance Raman spectroscopy to probe the population of native and misfolded cytochrome c on liposomes with 20 % tetraoleylcardiolipin (TOCL)/80 % dioleylphosphocholine (DOPC) and with 100 % TOCL as a function of TOCL concentration. Our data provided strong support for an earlier model, which predicts that the equilibrium between native and non-native conformations is shifted to the latter with decreasing protein occupation of liposomes.

129 - Cardiolipin Interactions with Cytochrome C increase Tyrosine Nitration Yields and Site-Specificity

Cytochrome c is a small protein that functions as carrier in the mitochondrial respiratory chain and pro-apoptotic mediator in the cytosol. This protein can also act as a peroxidase when exposed to high doses of hydrogen peroxide; when interacting with cardiolipin, a phospholipid from the inner mitochondrial membrane or after its oxidative post-translational modification by tyrosine nitration. Since mitochondria are a key place of intra cellular-generated peroxynitrite, the nitration of cytochrome c becomes a biologically-relevant event. In this work, we studied the effect of cardiolipin on cytochrome c tyrosine nitration and free radical formation when exposed to peroxynitrite, as well as the effect of a single residue, Y67, in the overall nitration. The effect of cardiolipin in cytochrome c exposed to peroxynitrite was studied by EPR analysis, showing an increase of free radicals when the protein was in presence of cardiolipin-containing liposomes. This increase of free radical formation was translated into an increase of tyrosine nitration, determined by western blot and proteomic analyses, in particular Y67. Also, studies were performed in order to establish the effect of nitration in the binding constant of cytochrome c with cardiolipin. Our results showed an increase of the binding constant when peroxynitrite was present. In order to determine the importance of Y67 in the reaction of peroxynitrite with cytochrome c in presence of cardiolipin, a mutant lacking this residue was used (Y67F). Notably, all differences observed using WT cyt c were abolished when using the Y67F mutant, suggesting that this residue, very close to the heme moiety of the protein, was responsible for the differences in reactivity and nitration observed. Also, when measuring the binding constant in presence of the Y67F mutant, no difference was observed upon addition of peroxynitrite to the reaction mixture. Our results support that a structural change induced by cardiolipin in cytochrome c ( i.e. weakening of the 6th coordination bond at the heme) increases tyrosine nitration yields, particularly in the heme-adjacent tyrosine 67, suggesting an iron-catalyzed event.

Cytochrome c reacts with cholesterol hydroperoxides to produce lipid- and protein-derived radicals.

Lipid peroxidation is a well-known process that has been implicated in many diseases. Recent evidence has shown that mitochondrial cholesterol levels are increased under specific conditions, making it an important target for peroxidation inside the mitochondria. Cholesterol peroxidation generates, as primary products, several hydroperoxides (ChOOH), which can react with transition metals and metalloproteins. In this sense, cytochrome c (CYTC), a heme protein largely found in the mitochondria, becomes a candidate to react with ChOOH. Using CYTC associated with SDS micelles to mimic mitochondrial conditions, we show that ChOOH induces dose-dependent CYTC Soret band bleaching, indicating that it is using ChOOH as a substrate. This reaction leads to protein oligomerization, suggesting the formation of a protein radical that, subsequently, recombines, giving dimers, trimers, and tetramers. EPR experiments confirmed the production of carbon-centered radicals from both protein and lipid in the presence of ChOOH. Similar results were obtained with linoleic acid hydroperoxides (LAOOH). In addition, replacing SDS micelles by cardiolipin-containing liposomes as the mitochondrial mimetic led to similar results with either ChOOH or LAOOH. Importantly, kinetic experiments show that CYTC bleaching is faster with ChOOH than with H2O2, suggesting that these hydroperoxides could be relevant substrates for CYTC peroxidase-like activity in biological media. Altogether, these results show that CYTC induces homolytic cleavage of lipid-derived hydroperoxides, producing lipid and protein radicals.

Coexistence of native-like and non-native partially unfolded ferricytochrome c on the surface of cardiolipin-containing liposomes.

Cytochrome c, in spite of adopting a rather rigid structure around its prosthetic heme group, is rather diverse with regard to its function and structural variability. On the surface of the inner membrane of mitochondria it serves as an electron transfer carrier. However, at conditions which have not yet been unambiguously identified, cytochrome c can adopt a variety of non-native conformations, some of which exhibit peroxidase activity. Cardiolipin-containing liposomes have served as ideal model system to investigate the various modes of interaction between cytochrome c and the inner mitochrondrial membrane. We probed the binding of horse heart ferricytochrome c to liposomes formed with 20% tetraoleoyl cardiolipin (TOCL) and 80% dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a function of lipid/protein ratio by fluorescence and visible circular dichroism spectroscopy. The obtained binding isotherms suggest that they reflect reversible binding processes, which excludes the possibility of significant protein insertion into the membrane. A global analysis of our data revealed the existence of two binding sites on the protein which causes rather different degrees of protein unfolding. We found that these two modes of interaction between protein and liposome led to conformational changes. While site 1 is relatively unaffected by NaCl, site 2 shows a more native-like state or a higher population of the native state in the presence of NaCl. At the highest utilized concentration of NaCl, there is only a 40% inhibition of the binding to site 2. We interpret our finding for this binding site as reflecting an equilibrium between electrostatically bound proteins with a high degree of unfolding and less unfolded proteins which bind either via H-bonding between lysine side chains and PO2(-) or hydrophobic interactions. With regard to site 2 binding, our results are reminiscent of the two-state equilibrium between a compact C and an extended E-state proposed by Pletneva and co-workers (Hanske et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 125-230). We conjecture that the nonelectrostatically bound proteins should have higher abilities to maintain the redox potential that is required for the function as an electron transfer protein.

Coexistence of Native-Like and Non-Native Misfolded Ferricytochrome C on the Surfac of Cardiolipin Containing Liposomes

Cytochrome c, in spite of adopting a rather rigid structure around its prosthetic heme group, is rather diverse with regard to its function and structural variability. On the surface of the inner membrane of mitochondria it serves as an electron transfer carrier. However, at conditions which have not yet been unambiguously identified, cytochrome c can adopt a variety of non-native conformations, some of which exhibit peroxidase activity. Cardiolipin-containing liposomes have served as ideal model system to investigate the various modes of interaction between cytochrome c and the inner mitochrondrial membrane. We probed the binding of horse heart ferricytochrome c to liposomes formed with 20% tetraoleoyl cardiolipin (TOCL) and 80% dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a function of lipid/protein ratio by fluorescence and visible circular dichroism spectroscopy. The obtained binding isotherms suggest that they reflect reversible binding processes, which excludes the possibility of significant prote...

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.

Structural insights into proapoptotic signaling mediated by MTCH2, VDAC2, TOM40 and TOM22

Mitochondrial Outer Membrane (MOM) Permeabilization (MOMP) is a critical step in the intrinsic pathway of apoptosis and is regulated by the Bcl-2 family of proteins. In vitro studies using cardiolipin-containing liposomes as a MOM model have suggested that a mitochondria-specific phospholipid, cardiolipin, is of crucial importance in MOMP. However, recently it has been found that the MOM contains much less cardiolipin than it is required for liposome permeabilization. Shortly thereafter, several MOM proteins, such as VDAC2, MTCH2, TOM22 and TOM40, have been identified as the Bax, Bak and tBid receptors that are indispensable in MOMP, but the underlying mechanisms are elusive. Here, proapoptotic signaling mediated by these MOM receptors was explored in terms of 3D-structures of interacting proteins using computational modeling. The formation under apoptotic conditions of the TOM40/TOM22/tBid protein complex possessing a fairly high binding affinity towards Bax is predicted, suggesting the recruitment of Bax to mitochondria by this complex in apoptotic cells. Our simulations predict the displacement of Bax from the TOM40/TOM22/tBid/Bax complex by another Bax in auto-catalytic manner and explain, in terms of structure, the tBid-mediated displacement of Bak from the VDAC2/Bak complex. Computational modeling revealed high-affinity binding of Bid to MTCH2 suggesting both a quasi-constitutive residence of Bid in MTCH2-bound state in healthy cells and its caspase-8-mediated cleavage there under apoptotic conditions. Overall, our results provide structural details for important stages of apoptotic signaling mediated by MOM receptors and enrich its mechanistic understanding.

Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10

The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies.

Dynamic Interaction of cBid with Detergents, Liposomes and Mitochondria

The BH3-only protein Bid plays a key role in the induction of mitochondrial apoptosis, but its mechanism of action is still not completely understood. Here we studied the two main activation events of Bid: Caspase-8 cleavage and interaction with the membrane bilayer. We found a striking reversible behaviour of the dissociation-association events between the Bid fragments p15 and p7. Caspase-8 cleavage does not induce per se separation of the two Bid fragments, which remain in a stable complex resembling the full length Bid. Detergents trigger a complete dissociation, which can be fully reversed by detergent removal in a range of protein concentrations from 100 µM down to 500 nM. Incubation of cBid with cardiolipin-containing liposomes leads to partial dissociation of the complex. Only p15 (tBid) fragments are found at the membrane, while p7 shows no tendency to interact with the bilayer, but complete removal of p7 strongly increases the propensity of tBid to become membrane-associated. Despite the striking structural similarities of inactive Bid and Bax, Bid does not form oligomers and reacts differently in the presence of detergents and membranes, highlighting clear differences in the modes of action of the two proteins. The partial dissociation of cBid triggered by the membrane is suggested to depend on the strong and specific interaction between p15 and p7. The reversible disassembly and re-assembly of the cBid molecules at the membrane was as well proven by EPR using spin labeled cBid in the presence of isolated mitochondria. The observed dynamic dissociation of the two Bid fragments could allow the assistance to the pore-forming Bax to occur repeatedly and may explain the proposed “hit-and-run" mode of action of Bid at the bilayer.

Ribose 5-Phosphate Glycation Reduces CytochromecRespiratory Activity and Membrane Affinity

Spontaneous glycation of bovine heart cytochrome c (cyt c) by the sugar ribose 5-phosphate (R5P) weakens the ability of the heme protein to transfer electrons in the respiratory pathway and to bind to membranes. Trypsin fragmentation studies suggest the preferential sites of glycation include Lys72 and Lys87/88 of a cationic patch involved in the association of the protein with its respiratory chain partners and with cardiolipin-containing membranes. Reaction of bovine cyt c with R5P (50 mM) for 8 h modified the protein in a manner that weakened its ability to transfer electrons to cytochrome oxidase by 60%. An 18 h treatment with R5P decreased bovine cyt c's binding affinity with cardiolipin-containing liposomes by an estimated 8-fold. A similar weaker binding of glycated cyt c was observed with mitoplasts. The reversal of the effects of R5P on membrane binding by ATP further supports an A-site modification. A significant decrease in the rate of spin state change for ferro-cyt c, thought to be due to cardiolipin insertion disrupting the coordination of Met to heme, was found for the R5P-treated cyt c. This change occurred to a greater extent than what can be explained by the permanent attachment of the protein to the liposome. Turbidity changes resulting from the multilamellar liposome fusion that is readily promoted by cyt c binding were not seen for the R5P-glycated cyt c samples. Collectively, these results demonstrate the negative impact that R5P glycation can have on critical electron transfer and membrane association functions of cyt c.

OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation

The dynamin-related GTPase OPA1 is mutated in autosomal dominant optic atrophy (DOA) (Kjer type), an inherited neuropathy of the retinal ganglion cells. OPA1 is essential for the fusion of the inner mitochondrial membranes, but its mechanism of action remains poorly understood. Here we show that OPA1 has a low basal rate of GTP hydrolysis that is dramatically enhanced by association with liposomes containing negative phospholipids such as cardiolipin. Lipid association triggers assembly of OPA1 into higher order oligomers. In addition, we find that OPA1 can promote the protrusion of lipid tubules from the surface of cardiolipin-containing liposomes. In such lipid protrusions, OPA1 assemblies are observed on the outside of the lipid tubule surface, a protein-membrane topology similar to that of classical dynamins. The membrane tubulation activity of OPA1 is suppressed by GTPγS. OPA1 disease alleles associated with DOA display selective defects in several activities, including cardiolipin association, GTP hydrolysis and membrane tubulation. These findings indicate that interaction of OPA1 with membranes can stimulate higher order assembly, enhance GTP hydrolysis and lead to membrane deformation into tubules.

Binding to and photo-oxidation of cardiolipin by the phthalocyanine photosensitizer Pc 4

Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Its peroxidation correlates with release of cytochrome c and induction of apoptosis. The phthalocyanine photosensitizer Pc 4 binds preferentially to the mitochondria and endoplasmic reticulum. Earlier Förster resonance energy transfer studies showed colocalization of Pc 4 and cardiolipin, which suggests cardiolipin as a target of photodynamic therapy (PDT) with Pc 4. Using liposomes as membrane models, we find that Pc 4 binds to cardiolipin-containing liposomes similarly to those that do not contain cardiolipin. Pc 4 binding is also studied in MCF-7c3 cells and those whose cardiolipin content was reduced by treatment with palmitate. Decreased levels of cardiolipin are quantified by thin-layer chromatography. The similar level of binding of Pc 4 to cells, irrespective of palmitate treatment, supports the lack of specificity of Pc 4 binding. Thus, factors other than cardiolipin are likely responsible for the preferential localization of Pc 4 in mitochondria. Nonetheless, cardiolipin within liposomes is readily oxidized by Pc 4 and light, yielding apparently mono- and dihydroperoxidized cardiolipin. If similar products result from exposure of cells to Pc 4-PDT, they could be part of the early events leading to apoptosis following Pc 4-PDT.

Interaction of the Tim44 C-Terminal Domain with Negatively Charged Phospholipids

The translocation of proteins from the cytosol into the mitochondrial matrix is mediated by the coordinated action of the TOM complex in the outer membrane, as well as the TIM23 complex and its associated protein import motor in the inner membrane. The focus of this work is the peripheral inner membrane protein Tim44. Tim44 is a vital component of the mitochondrial protein translocation motor that anchors components of the motor to the TIM23 complex. For this purpose, Tim44 associates with the import channel by direct interaction with the Tim23 protein. Additionally, it was shown in vitro that Tim44 associates with acidic model membranes, in particular those containing cardiolipin. The latter interaction was shown to be mediated by the carboxy-terminal domain of Tim44 [Weiss, C., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8890-8894]. The aim of this study was to determine the precise recognition site for negative lipids in the C-terminal domain of Tim44. In particular, we wanted to examine the recently suggested hypothesis that acidic phospholipids associate with Tim44 via a hydrophobic cavity that is observed in the high-resolution structure of the C-terminal domain of the protein [Josyula, R., et al. (2006) J. Mol. Biol. 359, 798-804]. Molecular dynamics simulations suggest that (i) the hydrophobic tail of lipids may interact with Tim44 via the latter's hydrophobic cavity and (ii) a region, located in the N-terminal alpha-helix of the C-terminal domain (helices A1 and A2), may serve as a membrane attachment site. To validate this assumption, N-terminal truncations of yeast Tim44 were examined for their ability to bind cardiolipin-containing phospholipid vesicles. The results indicate that removal of the N-terminal alpha-helix (helix A1) abolishes the capacity of Tim44 to associate with cardiolipin-containing liposomes. We suggest that helices A1 and A2, in Tim44, jointly promote the association of the protein with acidic phospholipids.