Background: BAK and BAX permeabilize the mitochondrial membrane during apoptosis. Results: Helices ␣2-␣5 of BAK form the "BH3-in-groove homodimer" in the membrane, which oligomerizes by juxtaposing the carboxyl termini of ␣3 and ␣5, respectively. Conclusion: A novel "␣3:␣3Ј, ␣5:␣5Ј oligomerization interface" exists in the BAK oligomeric pore. Significance: These results support a model for BAX/BAK pore formation, which constitutes a key regulatory step in mitochondrial apoptosis.

During apoptosis, the pro-apoptotic Bcl-2 family proteins BAK and BAX form large oligomeric pores in the mitochondrial outer membrane. Apoptotic factors, including cytochrome c, are released through these pores from the mitochondrial intermembrane space into the cytoplasm where they initiate the cascade of events leading to cell death. To better understand this pivotal step toward apoptosis, a method was developed to induce membrane permeabilization by BAK in the membrane without using the full-length protein. Using a soluble form of BAK with a hexahistidine tag at the C terminus and a liposomal system containing the Ni 2؉ -nitrilotriacetic acid lipid analog that can bind hexahistidine-tagged proteins, BAK oligomers were formed in the presence of the activator protein p7/p15Bid. In this system, we determined the conformational changes in BAK upon membrane insertion by applying the site-directed spin labeling method of EPR to 13 different amino acid locations. Upon membrane insertion, the BH3 domains were reorganized, and the ␣5-␣6 helical hairpin structure was partially exposed to the membrane environment. The monomer-monomer interface in the oligomeric structure was also mapped by measuring the distance-dependent spin-spin interactions for each residue location. Spin labels attached in the BH3 domain were juxtaposed within 5-10 Å distance in the oligomeric form in the membrane. These results are consistent with the current hypothesis that BAK or BAX forms homodimers, and these homodimers assemble into a higher order oligomeric pore. Detailed analyses of the data provide new insights into the structure of the BAX or BAK homodimer.

In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the ‘α3/α5’ interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known ‘α6:α6’ interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH.

The mitochondrial citrate transport protein (CTP) is critical to energy metabolism in eukaryotic cells. We demonstrate that 1,2,3-benzenetricarboxylate (BTC), the classic and defining inhibitor of the mitochondrial CTP, is a mixed inhibitor of the reconstituted Cys-less CTP, with a strong competitive component [i.e., a competitive inhibition constant (K ic ) of 0.12 Ϯ 0.02 mM and an uncompetitive inhibition constant (K iu ) of 3.04 Ϯ 0.74 mM]. Based on docking calculations, a model for BTC binding has been developed. We then determined the K ic values for each of the eight substrate binding site cysteine substitution mutants and observed increases of 62-to 261-fold relative to the Cys-less control, thereby substantiating the importance of each of these residues in BTC binding. It is noteworthy that we observed parallel increases in the K m for citrate transport with each of these binding site mutants, thereby confirming that with these CTP variants, K m approximates the K d (for citrate) and is therefore a measure of substrate affinity. To further substantiate the importance of these binding site residues, in silico screening of a database of commercially available compounds has led to discovery of the first purely competitive inhibitor of the CTP. Docking calculations indicate that this inhibitor spans and binds to both substrate sites simultaneously. Finally, we propose a kinetic model for citrate transport in which the citrate molecule sequentially binds to the external and internal binding sites (per CTP monomer) before transport.

The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [ 14 C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [ 14 C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.Citrate is a prominent intermediate in both carbohydrate and lipid metabolism. Once it is formed within the mitochondrial matrix as part of the tricarboxylic acid cycle, it is then either processed by the cycle enzymes to generate NADH and FADH 2 leading ultimately to the formation of ATP, or it can be transported out of the mitochondrial matrix across the inner membrane and into the intermembrane space via the mitochondrial inner membrane citrate transport protein (CTP) 2 (1, 2). Citrate then passively diffuses through a voltage-dependent anion-selective channel within the outer membrane, into the cytoplasm, where it is broken down by citrate lyase to acetylCoA and oxaloacetate. The resulting acetyl-CoA represents a prime carbon source fueling fatty acid, triacylglycerol, and sterol biosyntheses (3-6). Consequently, the CTP is critical to the energy metabolism of eukaryotic cells.The mitochondrial CTP catalyzes an obligatory exchange of the dibasic form of tricarboxylates (i.e. citrate and isocitrate) either for each other in yeast (7,8) or for dicarboxylates or phosphoenolpyruvate in higher eukaryotes (1, 2). Its function is altered in several diseases, including cancer (9) and diabetes (10), and it is thought to figure prominently in the abnormal bioenergetics observed with these pathologies. In terms of its function in the ...

The pro-apoptotic BCL-2 family protein BID activates BAK and/or BAX, which form oligomeric pores in the mitochondrial outer membrane. This results in the release of cytochrome c into the cytoplasm, initiating the apoptotic cascade. Here, we utilized liposomes encapsulating sulfo-rhodamine at a controlled temperature to improve upon a previously reported assay system with enhanced sensitivity and specificity for measuring membrane permeabilization by BID-dependent BAK activation. BAK activation was inhibited by BCL-X L protein but not by a mutant protein with impaired anti-apoptotic activity. With the assay system, we screened a chemical library and identified several compounds including trifluoperazine, a mitochondrial apoptosisinduced channel blocker. It inhibited BAK activation by direct binding to BAK and blocking the oligomerization of BAK.

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