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.

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.

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.

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.

Adenosine triphosphate synthesis coupled to K+ influx in mitochondria.

The influx of K+ into swollen mitochondria in the presence of valinomycin results in the synthesis of adenosine triphosphate in which approximately one H+ disappears per adenosine triphosphate synthesized. The synthesis is blocked by atractyloside but is insensitive to oligomycin and relatively insensitive to uncouplers.