MPP bound two mitochondrial import presequence peptides in extended conformations in a large polar cavity. The presequence conformations differ from the amphiphilic helical conformation recognized by mitochondrial import components. Our findings suggest that the presequences adopt context-dependent conformations through mitochondrial import and processing, helical for recognition by mitochondrial import machinery and extended for cleavage by the main processing component.

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We cloned a 38-kDa rat mitochondrial outer membrane protein (OM38) with structural homology to the central component of preprotein translocase of the fungal mitochondrial outer membrane, Tom40. Although it has no predictable ␣-helical transmembrane segments, OM38 is resistant to alkaline carbonate extraction and is inaccessible to proteases and polyclonal antibodies added from outside the mitochondria, suggesting that it is embedded in the membrane, probably in a ␤-barrel structure, as has been similarly speculated for fungal Tom40. Immunoprecipitation demonstrated that OM38 is associated with the major import receptors rTOM20 and rTOM22, and several other unidentified components with molecular masses of 5-10 kDa in digitoninsolubilized membrane: OM10, OM7.5, and OM5. Blue native polyacrylamide gel electrophoresis revealed that OM38 is a component of a ϳ400-kDa complex, firmly associating with rTOM22 and loosely associating with rTOM20. The preprotein in transit to the matrix interacted with the TOM complex containing OM38, and immunodepletion of OM38 resulted in the loss of preprotein import activity of the detergent-solubilized and reconstituted outer membrane vesicles. Taken together, these results indicate that OM38 is a structural and functional homolog of fungal Tom40 and functions as a component of the preprotein import machinery of the rat mitochondrial outer membrane.Most mitochondrial proteins are synthesized in the cytosol as preproteins, delivered to the mitochondrial surface by cytosolic factors such as hsp70 and mitochondrial import-stimulating factor, and transported to the intramitochondrial compartments by the preprotein import machinery of the outer and the inner membranes (the TOM 1 and TIM complexes, respectively)(1-4). The Saccharomyces cerevisiae TOM complex is composed of at least nine proteins (Tom71, -70, -40, -37, -22, -20, -7, -6, and -5) (5, 6). Tom40, the central component of the translocation channel, stably associates with the Tom22 receptor and small Tom components, Tom7, -6, and -5, and forms a ϳ400-kDa general insertion pore complex in yeast (5). Composition of the Neurospora crassa TOM complex is similar to that of S. cerevisiae, but Tom5 has yet to be identified in N. crassa. The mitochondrial inner membrane has two separate import machineries (7): the Tim23-Tim17 system and the Tim54-Tim22-Tim18 system. The Tim23-Tim17 system functions in the translocation of preproteins across the inner membrane in conjunction with Tim44, mhsp70, and GrpE (8 -10), whereas the Tim54-Tim22-Tim18 system functions in collaboration with the intermembrane space proteins Tim13, Tim12, Tim10, Tim9, and Tim8, in the import of proteins without a cleavable presequence, such as the phosphate carrier, the ADP/ATP carrier, and several Tim proteins (Tim23, Tim22, and Tim17) (10 -16).Although the fundamental mechanisms of mitochondrial protein import seem to be conserved from lower eukaryotes to mammals, only limited information is available for higher eukaryotic systems. Several mammalian counterparts have...

Bacillus thuringiensis crystal proteins, well known to be toxic to certain insects but not pathogenic to mammals, are used as insecticidal proteins in agriculture and forest management. We here identified a crystal protein that is non-insecticidal and non-hemolytic but has strong cytocidal activity against various human cells with a markedly divergent target specificity, e.g. highly cytotoxic to HepG2 and Jurkat and less cytotoxic to the normal hepatocyte (HC) and HeLa. In slices of liver and colon cancer tissues, the toxin protein preferentially killed the cancer cells, leaving other cells unaffected. The cytocidal effect of the protein is non-apoptotic with swelling and fragmentation of the susceptible cells, although the apoptotic process does occur when the cell damage proceeded slowly. The amino acid sequence deduced from the nucleotide sequence of the cloned gene of the protein has little sequence homology with the insecticidal crystal proteins of B. thuringiensis. These observations raise the presence of a new group of the B. thuringiensis toxin and the possibility of new applications for the protein in the medical field.

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.