The mitochondrial phospholipid metabolism critically depends on members of the conserved Ups1/PRELI-like protein family in the intermembrane space. Ups1 and Ups2 (also termed Gep1) were shown to regulate the accumulation of cardiolipin (CL) and phosphatidylethanolamine (PE), respectively, in a lipid-specific but coordinated manner. It remained enigmatic, however, how the relative abundance of both phospholipids in mitochondrial membranes is adjusted on the molecular level. Here, we describe a novel regulatory circuit determining the accumulation of Ups1 and Ups2 in the intermembrane space. Ups1 and Ups2 are intrinsically unstable proteins, which are degraded by distinct mitochondrial peptidases. The turnover of Ups2 is mediated by the i-AAA protease Yme1, whereas Ups1 is degraded by both Yme1 and the metallopeptidase Atp23. We identified Mdm35, a member of the twin Cx 9 C protein family, as a novel interaction partner of Ups1 and Ups2. Binding to Mdm35 ensures import and protects both proteins against proteolysis. Homologues to all components of this pathway are present in higher eukaryotes, suggesting that the regulation of mitochondrial CL and PE levels is conserved in evolution.

The generation of cellular energy depends on the coordinated assembly of nuclear and mitochondrial-encoded proteins into multisubunit respiratory chain complexes in the inner membrane of mitochondria. Here, we describe the identification of a conserved metallopeptidase present in the intermembrane space, termed Atp23, which exerts dual activities during the biogenesis of the F 1 F O -ATP synthase. On one hand, Atp23 serves as a processing peptidase and mediates the maturation of the mitochondrial-encoded F O -subunit Atp6 after its insertion into the inner membrane. On the other hand and independent of its proteolytic activity, Atp23 promotes the association of mature Atp6 with Atp9 oligomers. This assembly step is thus under the control of two substrate-specific chaperones, Atp10 and Atp23, which act on opposite sides of the inner membrane. Strikingly, both ATP10 and ATP23 were found to genetically interact with prohibitins, which build up large, ring-like assemblies with a proposed scaffolding function in the inner membrane. Our results therefore characterize not only a novel processing peptidase with chaperone activity in the mitochondrial intermembrane space but also link the function of prohibitins to the F 1 F O -ATP synthase complex. INTRODUCTIONMitochondria are dynamic organelles with essential catabolic and anabolic functions (Chan, 2006;McBride et al., 2006). Their most prominent function, the production of cellular energy, is carried out by multisubunit respiratory chain complexes in the inner membrane, whose formation depends on the coordinated assembly of mitochondrial and nuclear encoded subunits (Costanzo and Fox, 1990). A multitude of nuclear-encoded proteins has been identified, which assist the formation of these complexes at all stages, from the synthesis of mitochondrial-encoded core subunits and their membrane insertion, to their assembly within the inner membrane. This is exemplified by the F 1 F O -ATP synthase, which catalyzes the synthesis of ATP from ADP and organic phosphate using the electrochemical proton gradient across the inner membrane (Ackerman and Tzagoloff, 2005), but which has also a structural role for cristae morphology (Giraud et al., 2002). Core subunits of the proton-conducting F O -moiety of this enzyme are encoded within mitochondria and assemble with nuclear encoded subunits into functionally active F 1 F Oparticles. Efficient assembly is ensured by mitochondrial Hsp60 (Gray et al., 1990) and Hsp70 (Herrmann et al., 1994) and several substrate-specific chaperone proteins present within mitochondria. The latter include Atp11, Atp12, and Fmc1 (Ackerman and Tzagoloff, 1990;Lefebvre-Legendre et al., 2001), assembly factors of the matrix-exposed F 1 -ATPase particle, as well as Atp10, which promotes the assembly of mitochondrial-encoded Atp6 into the membrane-embedded F O -particle (Tzagoloff et al., 2004).Whereas these helper proteins assist the assembly of the F 1 F O -ATP synthase complexes in the inner membrane, a membrane-associated quality control system ensures t...

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