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.

No abstract