The mitochondrial protein apoptosis-inducing factor (AIF) translocates to the nucleus and induces apoptosis. Recent studies, however, have indicated the importance of AIF for survival in mitochondria. In the absence of a means to dissociate these two functions, the precise roles of AIF remain unclear. Here, we dissociate these dual roles using mitochondrially anchored AIF that cannot be released during apoptosis. Forebrain-specific AIF null (tel. AifDelta) mice have defective cortical development and reduced neuronal survival due to defects in mitochondrial respiration. Mitochondria in AIF deficient neurons are fragmented with aberrant cristae, indicating a novel role of AIF in controlling mitochondrial structure. While tel. AifDelta Apaf1(-/-) neurons remain sensitive to DNA damage, mitochondrially anchored AIF expression in these cells significantly enhanced survival. AIF mutants that cannot translocate into nucleus failed to induce cell death. These results indicate that the proapoptotic role of AIF can be uncoupled from its physiological function. Cell death induced by AIF is through its proapoptotic activity once it is translocated to the nucleus, not due to the loss of AIF from the mitochondria.

Nuclear-encoded proteins destined for insertion into the mitochondrial outer membrane, follow the same general pathway for import as proteins that are translocated to interior compartments within the organelle. This observation is true both for /?-barrel-type proteins and for proteins that contain hydrophobic a-helical transmembrane segments. In this review, we describe what is known about the various steps leading to protein insertion into the outer membrane, and discuss the energetics that favor vectorial translocation into and across this membrane. The selection of the outer membrane during import may involve a lateral release of the translocating polypeptide from the import machinery so that the appropriate domains of the protein become embedded in the lipid bilayer. One type of topogenic domain that can guarantee such selection of the outer membrane is a signal-anchor sequence of the type characterized for the bitopic protein Mas70p. It is suggested that a signal-anchor sequence selective for the mitochondrial outer membrane causes abrogation of polypeptide translocation and triggers the release of the transmembrane segment into the surrounding lipid bilayer, prior to any possibility for the commitment of translocation to the interior of the organelle. Specific structural features of the signal-anchor sequence specify its orientation in the membrane, and can confer on this sequence the ability to form homo-oligomers and hetero-oligomers. Strategies other than a signal-anchor sequence may be employed by other classes of proteins for selection of the outer-membrane. Of note is the ability of the outer-membrane import machinery to catalyze integration of the correct set of proteins into the outer-membrane bilayer, while allowing proteins that are destined for integration into the bilayer of the inner membrane to pass through unimpeded. Again, however, different proteins may employ different strategies. One model proposes that this can be accomplished by a combination of a matrix-targeting signal and a distal stop-transfer sequence. In this model, the formation of contact sites, which is triggered when the matrix-targeting signal engages the import machinery of the inner membrane, may prevent the outer-membrane translocon from recognizing and responding to the downstream stop-transfer domain. This allows the transmembrane segment to pass across the outer-membrane, and subsequently integrate into the inner membrane.Keywords. Mitochondria ; proteins, outer membrane ; import ; signal sequence anchor ; topology.The delimiting membrane of the mitochondrion, the outermembrane, typically contains 8-10% of the total protein of the organelle. These proteins include a diverse set of enzymes, pore structures, components of a complex protein recognition and import machinery, as yet undiscovered constituents of signal-transduction systems, and proteins that interact with the cytoskeleton and control organellar shape and movement. In many cases, the

This study was undertaken to identify novel mitochondrial membrane proteins that are potential targets for phosphorylation. Mitochondrial membranes were incubated in the presence of [y-32 P!ATP and the Triton X-114 extractable protein was subjected to ion-exchange and polyacrylamide gel chromatography to purify a major phosphorylated protein of approximately 17000 Da. The determined peptide sequence of the purified phosphoprotein corresponded to a segment of cytochrome c oxidase subunit IV, an inner membrane protein of 17160 Da. The identity of the phosphoprotein was confirmed by two-dimensional electrophoresis and Western blotting. The results identify mitochondrial cytochrome c oxidase subunit IV as a protein which is phosphorylated by an endogenous kinase.

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