The TIM10 chaperone facilitates the insertion of hydrophobic proteins at the mitochondrial inner membrane. Here we report the novel molecular mechanism of TIM10 assembly. This process crucially depends on oxidative folding in mitochondria and involves: (i) import of the subunits in a Cys-reduced and unfolded state; (ii) folding to an assembly-competent structure maintained by intramolecular disulfide bonding of their four conserved cysteines; and (iii) assembly of the oxidized zinc-devoid subunits to the functional complex. We show that intramolecular disulfide bonding occurs in vivo, whereas intermolecular disulfides observed in vitro are abortive intermediates in the assembly pathway. This novel mechanism of compartment-specific redox-regulated assembly is crucial for the formation of a functional TIM10 chaperone.Cysteine has unique biological functions by using its sulfhydryl (ϪSH) group in the active site of an enzyme, in chelating metals, or as the active site of disulfide reshuffling. For example, in the case of the molecular chaperone, Hsp33 activity is regulated by a redox switch with its inactive form reduced and zinc-coordinated and its active form turned on by oxidation and disulfide formation (1). The transcription factor OxyR is similarly activated through the formation of a disulfide bond and inactivated by enzymatic reduction with glutaredoxin (2). Disulfide bond formation in general is an essential step in the folding of many proteins, and it is catalyzed in vivo by the dsb system in the bacterial periplasm (3) and the functionally related PDI/Ero1 (4) system in the ER of eukaryotic cells. Although a mitochondrial intermembrane space sulfhydryl oxidase, Erv1p, has been identified (5), there has been no report suggesting disulfide bond formation in mitochondria. Here we demonstrate that the mitochondrial intermembrane space can allow oxidative folding events, challenging the commonly accepted notion that this compartment is in complete redox equilibrium with the reducing cytosol. We show that substrates for this oxidation event are Tim9 and Tim10, the subunits of the TIM10 chaperone that mediates hydrophobic protein insertion at the inner mitochondrial membrane (6 -9). This complex binds to the hydrophobic segments of the precursor (10) at an early import stage as the precursor emerges from the outer membrane protein import channel (translocase of the outer membrane, TOM 1 complex). Subsequently, the precursor is carried across the intermembrane space and passed onto the TIM22 membrane-embedded complex that facilitates insertion (11-13) through a twin pore involving two voltage-dependent steps (14).As all of the TIM subunits are imported themselves from the cytosol, correct assembly to their respective complex is essential for their function. Tim9 and Tim10 partner each other specifically to form the TIM10 complex, but the structural basis and the mechanism of this assembly process remain unclear. Although the "twin CX3C" motif common to all of the small Tim proteins is thought to be important for...

The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6–2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.

Apoptosis is accompanied by the activation of a number of apoptotic proteases (caspases) which selectively cleave speci®c cellular substrates. Caspases themselves are zymogens which are activated by proteolysis. It is widely believed that`initiator' caspases are recruited to and activated within apoptotic signalling complexes, and then cleave and activate downstream`eector' caspases. While activation of the eector caspase, caspase-3, has indeed been observed as distal to activation of several dierent initiator caspases, evidence for a further downstream proteolytic cascade is limited. In particular, there is little evidence that cellular levels of caspase-3 that are activated via one pathway are sucient to cleave and activate other initiator caspases. To address this issue, the ability of caspase-3, activated upon addition to cytosolic extracts of cytochrome c, to cause cleavage of caspase-2 was investigated. It was demonstrated that cleavage of caspase-2 follows, and is dependent upon, activation of caspase-3. Moreover, the activation of both caspases was inhibited by Bcl-2. Together, these data indicate that Bcl-2 can protect cells from apoptosis by acting at a point downstream from release of mitochondrial cytochrome c, thereby preventing a caspase-3 dependent proteolytic cascade.

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The TIM10 complex is localized in the mitochondrial intermembrane space and mediates insertion of hydrophobic proteins at the inner membrane. We have characterized TIM10 assembly and analyzed the structural properties of its subunits, Tim9 and Tim10. Both proteins are ␣-helical with a protease-resistant central domain, and each self-associates to form mainly dimers and trimers in solution. Tim9 and Tim10 bound to one another with submicromolar affinity in equimolar amounts and assembled in a stable, significantly extended complex that was indistinguishable from the native mitochondrial TIM10 complex. Importantly, the reconstituted TIM10 complex is functional because it bound to the physiological substrate ADP/ATP carrier and displayed chaperone activity in refolding the model substrate firefly luciferase. These data demonstrate that the individual subunits can exist as independent, dynamically self-associating proteins. Assembly into the thermodynamically stable hexameric complex is necessary for the TIM10 chaperone function.Almost all mitochondrial proteins are synthesized in the cytosol and then imported into the organelle in a process that is dictated by each protein's sequence and that is ensured by the function of specialized translocation machineries in the organelle (1-4). Most import and intramitochondrial sorting pathways are variants of the general "matrix pathway," first described in detail for matrix-targeted proteins (1-4). In this pathway, a mitochondrial precursor, which is usually synthesized with an N-terminal, positively charged, amphiphilic presequence, first interacts with cytosolic chaperones. It is then bound by a hetero-oligomeric receptor system on the surface of mitochondria in a process that requires ATP hydrolysis in the cytosol. The polypeptide chain is then transported across two hetero-oligomeric protein import channels, the TOM complex in the outer membrane and the TIM23 complex in the inner membrane. Translocation is completed by the electrophoretic function of the electrochemical potential across the inner membrane and the ATP-powered import motor attached to the inner side of the TIM23 complex. In this pathway, targeting of the polypeptide chain from one complex to the other appears to be directed by increasing avidity of the positive presequence for a series of acidic receptor domains ("acid chain hypothesis") (5-9).The structural basis of this mechanism is becoming increasingly clear through advances in understanding the structural characteristics of key components at different steps of this pathway. First, the solution structure (determined by NMR) of the cytosolic part of the Tom20 receptor in complex with a synthetic presequence has been solved (10, 11): this showed that the presequence is in ␣-helical conformation and that binding between the receptor and the presequence involves hydrophobic stretches. Second, the existence of a hydrophilic channel of TOM40 as the outer membrane import pore and its dynamic behavior have been established (12-16). Third, Tim23 was shown to f...

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Caspases, a family of specific proteases, have central roles in apoptosis [1]. Caspase activation in response to diverse apoptotic stimuli involves the relocalisation of cytochrome c from mitochondria to the cytoplasm where it stimulates the proteolytic processing of caspase precursors. Cytochrome c release is controlled by members of the Bcl-2 family of apoptosis regulators [2] [3]. The anti-apoptotic members Bcl-2 and Bcl-xL may also control caspase activation independently of cytochrome c relocalisation or may inhibit a positive feedback mechanism [4] [5] [6] [7]. Here, we investigate the role of Bcl-2 family proteins in the regulation of caspase activation using a model cell-free system. We found that Bcl-2 and Bcl-xL set a threshold in the amount of cytochrome c required to activate caspases, even in soluble extracts lacking mitochondria. Addition of dATP (which stimulates the procaspase-processing factor Apaf-1 [8] [9]) overcame inhibition of caspase activation by Bcl-2, but did not prevent the control of cytochrome c release from mitochondria by Bcl-2. Cytochrome c release was accelerated by active caspase-3 and this positive feedback was negatively regulated by Bcl-2. These results provide evidence for a mechanism to amplify caspase activation that is suppressed at several distinct steps by Bcl-2, even after cytochrome c is released from mitochondria.

CHN 2 ), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3,4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by twodimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities.