A myosin-Va tail fragment sequesters dynein light chains leading to apoptosis in melanoma cells.

Previous studies proposed that myosin-Va regulates apoptosis by sequestering pro-apoptotic Bmf to the actin cytoskeleton through dynein light chain-2 (DLC2). Adhesion loss or other cytoskeletal perturbations would unleash Bmf, allowing it to bind and inhibit pro-survival Bcl2 proteins. Here, we demonstrated that overexpression of a myosin-Va medial tail fragment (MVaf) harboring the binding site for DLC2 dramatically decreased melanoma cell viability. Morphological and molecular changes, including surface blebbing, mitochondrial outer membrane permeabilization, cytochrome-c and Smac release, as well as caspase-9/-3 activation and DNA fragmentation indicated that melanoma cells died of apoptosis. Immobilized MVaf interacted directly with DLCs, but complexed MVaf/DLCs did not interact with Bmf. Overexpression of DLC2 attenuated MVaf-induced apoptosis. Thus, we suggest that, MVaf induces apoptosis by sequestering DLC2 and DLC1, thereby unleashing the pair of sensitizer and activator BH3-only proteins Bmf and Bim. Murine embryonic fibroblasts (MEFs) lacking Bim and Bmf or Bax and Bak were less sensitive to apoptosis caused by MVaf expression than wild-type MEFs, strengthening the putative role of the intrinsic apoptotic pathway in this response. Finally, MVaf expression attenuated B16-F10 solid tumor growth in mice, suggesting that this peptide may be useful as an apoptosis-inducing tool for basic and translational studies.

Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis?

Apoptosis is a phenomenon fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other proapoptotic factors. Mitochondrial apoptosis-induced channel (MAC) is the mitochondrial apoptosis-induced channel and is responsible for cytochrome c release early in apoptosis. MAC activity is detected by patch clamping mitochondria at the time of cytochrome c release. The Bcl-2 family proteins regulate apoptosis by controlling the formation of MAC. Depending on cell type and apoptotic inducer, Bax and/or Bak are structural component(s) of MAC. Overexpression of the antiapoptotic protein Bcl-2 eliminates MAC activity. The focus of this review is a biophysical characterization of MAC activity and its regulation by Bcl-2 family proteins, and ends with some discussion of therapeutic targets.

A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast.

During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.

BH3 death domain peptide induces cell type-selective mitochondrial outer membrane permeability.

The BH3 domain is essential for the release of cytochrome c from mitochondria by pro-apoptotic Bcl-2 family proteins during apoptosis. This study tested the hypothesis that a Bax peptide that includes the BH3 domain can permeabilize the mitochondrial outer membrane and release cytochrome c in the absence of a permeability transition at the mitochondrial inner membrane. BH3 peptide (0.1-60 microm) released cytochrome c from mitochondria in the presence of physiological concentrations of ions in a cell type-selective manner, whereas a BH3 peptide with a single amino acid substitution was ineffective. The release of cytochrome c by BH3 peptide correlated with the presence of endogenous Bax at the mitochondria and its integral membrane insertion. Cytochrome c release was accompanied by adenylate kinase release, was not associated with mitochondrial swelling or substantial loss of electrical potential across the inner membrane, and was unaffected by inhibitors of the permeability transition pore. Cytochrome c release was, however, inhibited by Bcl-2. Although energy-coupled respiration was inhibited after the release of cytochrome c, mitochondria maintained membrane potential in the presence of ATP due to the reversal of the ATP synthase. Overall, results support the hypothesis that BH3 peptide releases cytochrome c by a Bax-dependent process that is independent of the mitochondrial permeability transition pore but regulated by Bcl-2.

Mitochondrial precursor signal peptide induces a unique permeability transition and release of cytochrome c from liver and brain mitochondria.

This study tested the hypothesis that mitochondrial precursor targeting peptides can elicit the release of cytochrome c from both liver and brain mitochondria by a mechanism distinct from that mediated by the classical, Ca2+-activated permeability transition pore. Human cytochrome oxidase subunit IV signal peptide (hCOXIV1-22) at concentrations from 15 to 100 microM induced swelling, a decrease in membrane potential, and cytochrome c release in both types of mitochondria. Although cyclosporin A and bongkrekic acid were without effect, dibucaine, propanolol, dextran, and the uncoupler FCCP were each able to inhibit signal peptide-induced swelling and cytochrome c release. Adenylate kinase was coreleased with cytochrome c, arguing against a signal peptide-induced cytochrome c-specific pathway of efflux across the outer membrane. Taken together, the data indicate that a human mitochondrial signal peptide can evoke the release of cytochrome c from both liver and brain mitochondria by a unique permeability transition that differs in several characteristics from the classical mitochondrial permeability transition.

Overexpression of Bcl-2 suppresses the calcium activation of a mitochondrial megachannel.

The molecular mechanism(s) by which Bcl-2 regulates apoptosis is poorly understood. Bcl-2 suppresses apoptosis by inhibiting calcium activation of the permeability transition of mitochondria. In this patch-clamp study, overexpression of Bcl-2 in mitochondria of cultured cells suppressed calcium activation of a high conductance channel that may underlie the permeability transition. All other single channel parameters were identical when multiple conductance channel activities of mitochondria from control and Bcl-2 overexpressing cells were compared. Bcl-2 forms channels in artificial membranes; however, no novel channel activities could be linked to Bcl-2 overexpression, suggesting Bcl-2 does not form channels in native inner membranes of mitochondria.

MCC and PSC, the putative protein import channels of mitochondria.

All but a small fraction of the hundreds of proteins in a mitochondrion are synthesized in the cytoplasm and imported into the organelle. Water-filled channels are integral to the process of translocating proteins since channels can provide an aqueous pathway through the hydrophobic environment of the membrane. The MCC (multiple conductance channel) and PSC (peptide-sensitive channel) are two high-conductance channels previously identified in electrophysiological studies of mitochondrial membranes. MCC and PSC are the putative pores of the import complexes of the inner and outer membranes, respectively. The genetic, biochemical, and biophysical evidence regarding these assignments are summarized herein. These findings support the identification of MCC and PSC as the protein import channels of mitochondria.

Signal presequences increase mitochondrial permeability and open the multiple conductance channel.

We have reported that the signal presequence of cytochrome oxidase subunit IV from Neurospora crassa increases the permeability of isolated rat liver mitochondria [P. M. Sokolove and K. W. Kinnally (1996) Arch. Biochem. Biophys. 336, 69] and regulates the behavior of the mutiple conductance channel (MCC) of yeast inner mitochondrial membrane [T. A. Lohret and K. W. Kinnally (1995) J. Biol. Chem. 270, 15950]. Here we examine in greater detail the action of a number of mitochondrial presequences from various sources and of several control peptides on the permeability of isolated rat liver mitochondria and on MCC activity monitored via patch-clamp techniques in both mammalian mitoplasts and a reconstituted yeast system. The data indicate that the ability to alter mitochondrial permeability is a property of most, but not all, signal peptides. Furthermore, it is clear that, although signal peptides are characterized by positive charge and the ability to form amphiphilic alpha helices, these two characteristics are not sufficient to guarantee mitochondrial effects. Finally, the results reveal a strong correlation between peptide effects on the permeability of isolated mitochondria and on MCC activity: peptides that induced swelling of mouse and rat mitochondria also activated the quiescent MCC of mouse mitoplasts and induced flickering of active MCC reconstituted from yeast mitochondrial membranes. Moreover, relative peptide efficacies were very similar for mitochondrial swelling and both types of patch-clamp experiments. We propose that patch-clamp recordings of MCC activity and the high-amplitude swelling induced by signal peptides reflect the opening of a single channel. Based on the selective responsiveness of that channel to signal peptides and the dependence of its opening in isolated mitochondria on membrane potential, we further suggest that the channel is involved in the mitochondrial protein import process.

Bacterial expression and characterization of the mitochondrial outer membrane channel. Effects of n-terminal modifications.

Several forms of the voltage-dependent anion-selective channel (VDAC) have been expressed at high yield in Escherichia coli. Full-length constructs of the proteins of Neurospora crassa and Saccharomyces cerevisiae (ncVDAC and scVDAC) have been made with 20-residue-long, thrombin-cleavable, His6-containing N-terminal extensions. ncVDAC purified from bacteria or mitochondria displays a far-UV CD spectrum (in 1% lauryl dimethylamine oxide at pH 6-8) similar to that of bacterial porins, indicating extensive beta-sheet structure. Under the same conditions, the CD spectrum of bacterially expressed scVDAC indicates lower beta-sheet content, albeit higher than that of mitochondrial scVDAC under the same conditions. In phospholipid bilayers, the bacterially expressed proteins (with or without N-terminal extensions) form typical VDAC-like channels with stable, large conductance open states (4-4.5 nanosiemens in 1 M KCl) and voltage-dependent transitions to a predominant substate (about 2 nanosiemens). A variant of scVDAC missing the first eight residues and having no N-terminal extension also has been expressed in E. coli. The truncated protein has a CD spectrum similar to that of mitochondrial scVDAC, but its channel activity is abnormal, exhibiting an unstable open state and rapid transitions between multiple subconductance levels.

Two high conductance channels of the mitochondrial inner membrane are independent of the human mitochondrial genome.

Patch-clamp techniques were used to characterize the channel activity of mitochondrial inner membranes of two human osteosarcoma cell lines: a mitochondrial genome-deficient (rho0) line and its corresponding parental (rho+) line. Previously, two high conductance channels, mitochondrial Centum picoSiemen (mCS) and multiple conductance channels (MCC), were detected in murine mitochondria. While MCC was assigned to the protein import in yeast mitochondria, the role of mCS is unknown. This study demonstrates that mCs and MCC activities from mouse mitochondria are indistinguishable from those of human mitochondria. The channel activities and their functional expression levels are not altered in cells lacking mtDNA. Hence, rho0 cells may provide a model system for elucidating the role of mitochondrial channels in disease processes and apoptosis.

Effects of carbonyl cyanide phenylhydrazones on two mitochondrial ion channel activities.

The respiratory uncouplers carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) affect the activities of two mitochondrial ion channels from mouse liver. At micromolar concentrations, the phenylhydrazones block the voltage-dependent 100-pS channel, mCS, and induce the multiple-conductance-level channel, MCC. The binding site(s) involved in perturbation of channel activities are probably distinct from the sites involved in uncoupling of oxidative phosphorylation which occurs at nanomolar concentrations of the phenylhydrazones. The effects of FCCP and CCCP on the mitochondrial ion channels could be partially reversed by washing with fresh media and were always reversed by perfusion with dithiothreitol. These results indicate that the effects of the phenylhydrazones on mitochondrial ion channels may be related to the ability of these compounds to act as sulfhydryl reagents and not to their protonophoric and uncoupling activity.

Tim23, a protein import component of the mitochondrial inner membrane, is required for normal activity of the multiple conductance channel, MCC.

We previously showed that the conductance of a mitochondrial inner membrane channel, called MCC, was specifically blocked by peptides corresponding to mitochondrial import signals. To determine if MCC plays a role in protein import, we examined the relationship between MCC and Tim23p, a component of the protein import complex of the mitochondrial inner membrane. We find that antibodies against Tim23p, previously shown to inhibit mitochondrial protein import, inhibit MCC activity. We also find that MCC activity is altered in mitochondria isolated from yeast carrying the tim23-1 mutation. In contrast to wild-type MCC, we find that the conductance of MCC from the tim23-1 mutant is not significantly blocked by mitochondrial presequence peptides. Tim23 antibodies and the tim23-1 mutation do not, however, alter the activity of PSC, a presequence-peptide sensitive channel in the mitochondrial outer membrane. Our results show that Tim23p is required for normal MCC activity and raise the possibility that precursors are translocated across the inner membrane through the pore of MCC.

The mitochondrial protein import pathway: are precursors imported through membrane channels?

Mitochondrial biogenesis requires the import of hundreds of different proteins from the cytosol. Protein import into mitochondria is a multistep pathway that includes recognition of precursor proteins by machinery both in the cytoplasm and on the mitochondrial surface, translocation of the precursor across one or both mitochondrial membranes, and folding of the protein after its import into the organelle. Over the past several years, many components of the import machinery have been identified using both biochemical and genetic methods. Recently, significant progress has been made determining the function of some of these import proteins. One purpose of this minireview is to summarize our current understanding of the import pathway, and to introduce the topics of the minireviews that will follow. The other goal of this minireview is to discuss recent findings suggesting that proteins are translocated across both the mitochondrial inner and outer membranes through aqueous channels.

A mitochondrial signal peptide from Neurospora crassa increases the permeability of isolated rat liver mitochondria.

Mitochondria that contain Ca2+ can be induced by a variety of triggering agents and conditions to undergo a permeability transition (PT); the inner membrane becomes nonselectively permeable to small solutes. Mastoparan, an amphipathic peptide from wasp venom, has recently been reported to induce this transition (Pfeiffer et al., 1995, J. Biol. Chem. 270,4923). We have examined the effect on the permeability of isolated rat liver mitochondria of a second amphipathic peptide, the signal sequence of cytochrome oxidase subunit IV from Neurospora crassa (pCoxIV, amino acids 3-22), which targets subunit IV to its mitochondrial location. Permeability increases were visualized via mitochondrial swelling with the following results. (1) pCoxIV (5-100 microM) induced concentration-dependent mitochondrial swelling. Control peptides from the N- and C-termini of the voltage-dependent anion-selective channel had no such effect. (2) Swelling required mitochondrial energization; it was eliminated or halted by the uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. (3) Peptide-induced swelling was slowed by increasing concentrations of KCl. (4) Swelling was enhanced by inorganic phosphate (<1 mM). (5) Trifluoperazine (50 microM), propranolol (0.5 mM), and dibucaine (0.5 mM) were potent inhibitors of peptide-induced swelling, whereas other inhibitors of the classical PT (cyclosporin A, EGTA, and ADP) inhibited only partially. (6) pCoxIV opened a pore rather than disrupting mitochondrial membrane structure, but 50% inhibition of peptide-induced swelling required polyethylene glycol of molecular weight substantially larger than that needed to inhibit the Ca2+-induced PT to the same extent. In summary, pCoxIV opens a pore in isolated mitochondria. The dependence of pore opening on membrane potential and the inhibition of the peptide-induced permeability increase by increasing salt concentration suggest that this effect of the signal peptide is related to its interactions with mitochondria during protein import. The peptide-induced pore appears, however, to be distinct from both the classical permeability transition pore and the mastoparan-induced permeability increase.

Circular dichroism studies of the mitochondrial channel, VDAC, from Neurospora crassa.

The protein that forms the voltage-gated channel VDAC (or mitochondrial porin) has been purified from Neurospora crassa. At room temperature and pH 7, the circular dichoism (CD) spectrum of VDAC suspended in octyl beta-glucoside is similar to those of bacterial porins, consistent with a high beta-sheet content. When VDAC is reconstituted into phospholipid liposomes at pH 7, a similar CD spectrum is obtained and the liposomes are rendered permeable to sucrose. Heating VDAC in octyl beta-glucoside or in liposomes results in thermal denaturation. The CD spectrum irreversibly changes to one consistent with total loss of beta-sheet content, and VDAC-containing liposomes irreversibly lose sucrose permeability. When VDAC is suspended at room temperature in octyl beta-glucoside at pH < 5 or in sodium dodecyl sulfate at pH 7, its CD spectrum is consistent with partial loss of beta-sheet content. The sucrose permeability of VDAC-containing liposomes is decreased at low pH and restored at pH 7. Similarly, the pH-dependent changes in the CD spectrum of VDAC suspended in octyl beta-glucoside also are reversible. These results suggest that VDAC undergoes a reversible conformational change at low pH involving reduced beta-sheet content and loss of pore-forming activity.

Perspectives on the mitochondrial multiple conductance channel.

A multiple conductance channel (MCC) with a peak conductance of over 1 nS is recorded from mitoplasts (mitochondria with the inner membrane exposed) using patch-clamp techniques. MCC shares many general characteristics with other intracellular megachannels, many of which are weakly selective, voltage-dependent, and calcium sensitive. A role in protein import is suggested by the transient blockade of MCC by peptides responsible for targeting mitochondrial precursor proteins. MCC is compared with the peptide-sensitive channel of the outer membrane because of similarities in targeting peptide blockade. The pharmacology and regulation of MCC by physiological effectors are reviewed and compared with the properties of the pore hypothesized to be responsible for the mitochondrial inner membrane permeability transition.

Activity of the mitochondrial multiple conductance channel is independent of the adenine nucleotide translocator.

The functional relationship between the adenine nucleotide translocator (ANT) and the mitochondrial multiple conductance channel (MCC) was investigated using patch-clamp techniques. MCC activity with the same conductance, ion selectivity, voltage dependence, and peptide sensitivity could be reconstituted from inner membrane fractions derived from mitochondria of ANT-deficient and wild-type Saccharomyces cerevisiae. In addition, the MCC activity of mouse kidney mitoplasts was unaffected by carboxyatractyloside, a known inhibitor of ANT and inducer of a permeability transition. These results suggest that MCC activity is independent of ANT.

Targeting peptides transiently block a mitochondrial channel.

The effects of synthetic targeting peptides on the activity of the multiple conductance channel (MCC) of mouse and yeast mitochondria were investigated using patch-clamp techniques. Amino-terminal targeting peptides of two inner membrane proteins reversibly decreased the open probability and mean open time of MCC. One of these targeting peptides had no effect on two other voltage-dependent mitochondrial channels. Furthermore, the effects induced by the two targeting peptides on MCC were not elicited by two peptides of an outer membrane protein. The specific interactions of targeting peptides with MCC suggest that this channel may be involved in protein import across the inner mitochondrial membrane.

Multiple conductance channel activity of wild-type and voltage-dependent anion-selective channel (VDAC)-less yeast mitochondria.

Yeast mitoplasts (mitochondria with the outer membrane stripped away) exhibit multiple conductance channel activity (MCC) in patch-clamp experiments that is very similar to the activity previously described in mammalian mitoplasts. The possible involvement of the voltage-dependent anion-selective channel (VDAC) of the outer membrane in MCC activity was explored by comparing the channel activity in wild-type yeast mitoplasts with that of a VDAC-deletion mutant. The channel activity recorded from the mutant is essentially the same as that of the wild-type in the voltage range of -40 to 30 mV. These observations indicate that VDAC is not required for MCC activity. Interestingly, the channel activity of the VDAC-less yeast mitoplasts exhibits altered gating properties at transmembrane potentials above and below this range. We conclude that the deletion of VDAC somehow results in a modification of MCC's voltage dependence. In fact, the voltage profile recorded from the VDAC-less mutant resembles that of VDAC.

Single-channel activity induced in mitoplasts by alkaline pH.

Exposure of patch-clamped mitoplasts to alkaline pH induces a reversible conductance increase (Antonenko, Yu. N., Kinnally, K.W. and Tedeschi, H. (1991) J. Membr. Biol. 124, 151-158) which is due to an increase in open probability of a channel activity of 15 pS and larger transitions. The present study defines in more detail some of the characteristics of the channel activity involved in this conductance increase. The results suggest the presence of two channels one slightly cation-selective of approx. 15 pS (referred to here as alkaline-induced cation-selective activity, ACA) and another slightly anion selective of approx. 45 pS (referred to as alkaline-induced anion-selective activity, AAA). The possible implication of these results in relation to other channels and the permeability transitions reported by others using mitochondrial suspensions is discussed.