The gene encoding the yeast mitochondrial outer membrane channel VDAC was subjected to site-directed mutagenesis to change amino acids at 29 positions to residues differing in charge from the wild-type sequence. The mutant genes were then expressed in yeast, and the physiological consequences of single and multiple amino acid changes were assessed after isolation and insertion of mutant channels into phospholipid bilayers. Selectivity changes were observed at 14 sites distributed throughout the length of the molecule. These sites are likely to define the position of the protein walls lining the aqueous pore and hence, the transmembrane segments. These results have been used to develop a model of the open state of the channel in which each polypeptide contributes 12 beta strands and one alpha helix to form the aqueous transmembrane pathway.

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The BCL-2 family includes both proapoptotic (e.g., BAX and BAK) and antiapoptotic (e.g., BCL-2 and BCL-X L ) molecules. The cell death-regulating activity of BCL-2 members appears to depend on their ability to modulate mitochondrial function, which may include regulation of the mitochondrial permeability transition pore (PTP). We examined the function of BAX and BCL-X L using genetic and biochemical approaches in budding yeast because studies with yeast suggest that BCL-2 family members act upon highly conserved mitochondrial components. In this study we found that in wild-type yeast, BAX induced hyperpolarization of mitochondria, production of reactive oxygen species, growth arrest, and cell death; however, cytochrome c was not released detectably despite the induction of mitochondrial dysfunction. Coexpression of BCL-X L prevented all BAX-mediated responses. We also assessed the function of BCL-X L and BAX in the same strain of Saccharomyces cerevisiae with deletions of selected mitochondrial proteins that have been implicated in the function of BCL-2 family members. BAX-induced growth arrest was independent of the tested mitochondrial components, including voltage-dependent anion channel (VDAC), the catalytic ␤ subunit or the ␦ subunit of the F 0 F 1 -ATP synthase, mitochondrial cyclophilin, cytochrome c, and proteins encoded by the mitochondrial genome as revealed by [rho 0 ] cells. In contrast, actual cell killing was dependent upon select mitochondrial components including the ␤ subunit of ATP synthase and mitochondrial genome-encoded proteins but not VDAC. The BCL-X L protection from either BAX-induced growth arrest or cell killing proved to be independent of mitochondrial components. Thus, BAX induces two cellular processes in yeast which can each be abrogated by BCL-X L : cell arrest, which does not require aspects of mitochondrial biochemistry, and cell killing, which does.

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro–imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (⌬por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37°C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of ⌬por1 yeast grown on glycerol at 37°C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in ⌬por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30°C, albeit more slowly than ⌬por1 single mutants. Like ⌬por1 single mutants, they could not grow on glycerol at 37°C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, ⌬por1 cells, ⌬por1 ⌬por2 cells, and ⌬por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in ⌬por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37°C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.

Background: TSPO has been proposed to be a critical regulator of the permeability transition pore (PTP). Results: TSPO-null mitochondria and cardiac tissue show no difference from controls in pore function, response to ligands, or response to ischemia/reperfusion injury. Conclusion: Regulation of the PTP by the outer membrane must rely on unknown proteins. Significance: Our results call into question studies implicating TSPO in pathological processes through the PTP.

Voltage-gated ion-channel proteins contain "voltage-sensing" domains that drive the conformational transitions between open and closed states in response to changes in transmembrane voltage. We have used site-directed mutagenesis to identify residues affecting the voltage sensitivity of a mitochondrial channel, the voltage-dependent anion-selective channel (VDAC). Although charge changes at many sites had no effect, at other sites substitutions that increased positive charge also increased the steepness of voltage dependance and substitutions that decreased positive charge decreased voltage dependance by an appropriate amount. In contrast to the plasma membrane K+ and Na+ channels, these residues are distributed over large parts of the VDAC protein. These results have been used to define the conformational transitions that accompany voltage gating of an ion channel. This gating mechanism requires the movement of large portions of the VDAC protein through the membrane.Voltage-dependent membrane channels change their ability to conduct ions in response to small changes in membrane electric potential. This sensitivity to voltage is thought to result from the linkage of the conformational change responsible for the change in ion conductance and the movement of a charged domain through the membrane potential or the effective alignment of a large dipole with respect to the electric field. The charged domain or region containing the large dipole would then correspond to the "voltage sensor." For the best studied plasma membrane channels, the voltagesensitive K+ and Na+ channels, a localized region of the protein, the S4 domain, has been identified by site-directed mutagenesis as containing residues forming the voltage sensor of these channels (1-6). Despite this information, a true molecular picture of the transitions that occur in response to voltage changes to cause these channels to open and close has been and will be difficult to define experimentally since the channels are formed by large oligomeric proteins. Smaller channels that form voltage-gated pores with much less protein are likely to provide insights into how an electric field can induce these molecular transitions. We have used sitedirected mutations to identify the sensor region in one such channel, a mitochondrial channel, the voltage-dependent anion-selective channel (VDAC). In contrast to the S4 domain identified in Na+ and K+ channels, the voltage-sensing domain in VDAC is distributed over a relatively large region of the protein. Our data support a mechanism for voltage gating of this channel that requires the movement of large amounts of protein mass through the membrane. MATERIALS AND METHODSVDAC genes were modified by site-directed mutagenesis and introduced into a Saccharomyces cerevisiae strain lackingThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. the chromosomal copy of the V...

The outer membrane of mitochondria contains channels called VDAC (mitochondrial porin), which are formed by a single 30-kDa protein. Cysteine residues introduced by site-directed mutagenesis at sites throughout Neurospora crassa VDAC (naturally devoid of cysteine) were specifically biotinylated prior to reconstitution into planar phospholipid membranes. From previous studies, binding of streptavidin to single biotinylated sites results in one of two effects: reduced single-channel conductance without blockage of voltage gating (type 1) or locking of the channels in a closed conformation (type 2). All sites react with streptavidin only from one side of the membrane. Here, we extend this approach to VDAC molecules containing two cysteines and determine the location of each biotinylated residue with respect to the other within the membrane. When a combination of a type 1 and a type 2 site was used, each site could be observed to react with streptavidin. Two sets of sites located on opposite surfaces of the membrane were identified, thereby establishing the transmembrane topology of VDAC. A revised folding pattern for VDAC, consisting of 1 ␣ helix and 13 ␤ strands, is proposed by combining these results with previously obtained information on which sites are lining the aqueous pore.VDAC channels are found in the mitochondrial outer membrane of cells from all eukaryotic kingdoms (1). They are the major pathways by which metabolites, including ATP, pass through this outer membrane (2, 3) and, therefore, are likely to play important roles in the regulation of mitochondrial functions.Each VDAC channel consisting of a single 30-kDa polypeptide (4, 5) forms an aqueous pore ϳ3 nm in diameter (6, 7). Detailed information on the voltage-gating properties of VDAC and the associated conformational rearrangements have been obtained from studies of channels reconstituted into planar phospholipid membranes. These studies have demonstrated that, in response to transmembrane potentials of 30 mV or above, part of the channel wall, which serves as the voltage sensor, moves out of the membrane resulting in channel closure (8 -10). These closed channels are essentially impermeable to ATP and have a reduced permeability to organic anions (2, 11). However, they are still permeable to small non-electrolytes (up to 1.8 nm in diameter) and to small monovalent cations (12). VDAC can close in response to transmembrane potentials of both polarities, due to the presence of two different gating processes.The transmembrane topology of VDAC molecules has yet to be firmly established. Theoretical considerations predicted a "␤-barrel" structure (13,14). "Sided" ␤ strands that would be appropriate to separate an apolar from a polar environment, were tested by the use of site-directed mutations (15). If engineered charge changes in proposed transmembrane segments affected channel selectivity, the associated protein segment was proposed to be a transmembrane strand. A lack of effect resulted in assignment of the segment to the membrane surface. In thes...