f Mitochondria possess an outer membrane (OMM) and an inner membrane (IMM), which folds into invaginations called cristae. Lipid composition, membrane potential, and proteins in the IMM influence organization of cristae. Here we show an essential role of the OMM protein Sam50 in the maintenance of the structure of cristae. Sam50 is a part of the sorting and assembly machinery (SAM) necessary for the assembly of ␤-barrel proteins in the OMM. We provide evidence that the SAM components exist in a large protein complex together with the IMM proteins mitofilin and CHCHD3, which we term the mitochondrial intermembrane space bridging (MIB) complex. Interactions between OMM and IMM components of the MIB complex are crucial for the preservation of cristae. After destabilization of the MIB complex, we observed deficiency in the assembly of respiratory chain complexes. Long-term depletion of Sam50 influences the amounts of proteins from all large respiratory complexes that contain mitochondrially encoded subunits, pointing to a connection between the structural integrity of cristae, assembly of respiratory complexes, and/or the maintenance of mitochondrial DNA (mtDNA).

Voltage-dependent anion-selective channel (VDAC) is a b-barrel protein in the outer mitochondrial membrane that is necessary for metabolite exchange with the cytosol and is proposed to be involved in certain forms of apoptosis. We studied the biogenesis of VDAC in human mitochondria by depleting the components of the mitochondrial import machinery by using RNA interference. Here, we show the importance of the translocase of the outer mitochondrial membrane (TOM) complex in the import of the VDAC precursor. The deletion of Sam50, the central component of the sorting and assembly machinery (SAM), led to both a strong defect in the assembly of VDAC and a reduction in the steadystate level of VDAC. Metaxin 2-depleted mitochondria had reduced levels of metaxin 1 and were deficient in import and assembly of VDAC and Tom40, but not of three matrix-targeted precursors. We also observed a reduction in the levels of metaxin 1 and metaxin 2 in Sam50-depleted mitochondria, implying a connection between these three proteins, although Sam50 and metaxins seemed to be in different complexes. We conclude that the pathway of VDAC biogenesis in human mitochondria involves the TOM complex, Sam50 and metaxins, and that it is evolutionarily conserved.

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨm). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨm. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨm loss and apoptosis, demonstrating that dissipation of ΔΨm is a requirement for cell death caused by neisserial infection.

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