The yeast mitochondrial Oxa1 protein is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1 mediates the insertion of proteins (nuclearly and mitochondrially encoded) into the inner membrane. The mitochondrially encoded substrates interact directly with Oxa1 during their synthesis as nascent chains and in a manner that is supported by the associated ribosome. We have investigated if the Oxa1 complex interacts with the mitochondrial ribosome. Evidence to support a physical association between Oxa1 and the large ribosomal subunit is presented. Our data indicate that the matrix‐exposed C‐terminal region of Oxa1 plays an important role supporting the ribosomal–Oxa1 interaction. Truncation of this C‐terminal segment compromises the ability of Oxa1 to support insertion of substrate proteins into the inner membrane. Oxa1 can be cross‐linked to Mrp20, a component of the large ribosomal subunit. Mrp20 is homologous to L23, a subunit located next to the peptide exit tunnel of the ribosome. We propose that the interaction of Oxa1 with the ribosome serves to enhance a coupling of translation and membrane insertion events.

The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc 1 )-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc 1 -COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc 1 -COX supercomplex is perturbed, whereby a decrease in the III 2 -IV 2 assembly state relative to the III 2 -IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc 1 -COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc 1 -COX-TIM23 supercomplex are also discussed. INTRODUCTIONATP synthesized within the mitochondrial matrix by the F 1 F o -ATP synthase is transported out across the inner membrane by the ADP/ATP carrier (AAC) proteins, a process that is coupled to the import of ADP into the mitochondrial matrix (Klingenberg, 1989;Gawaz et al., 1990;Drgon et al., 1991;Pebay-Peyroula and Brandolin, 2004;Nury et al., 2006). The AAC proteins span the inner mitochondrial membrane six times with short N-and C-terminal hydrophilic tails in the intermembrane space (Pebay-Peyroula et al., 2003). A number of mitochondrial AAC isoforms have been shown to exist in many organisms. In the yeast Saccharomyces cerevisiae, the model organism of this study, there are three AAC isoforms, encoded by the AAC1, AAC2, and AAC3 genes (Klingenberg, 1989;Gawaz et al., 1990;Drgon et al., 1991). In aerobically grown cells, the Aac2 protein is the most abundant AAC isoform and the Aac1 represents a minor isoform. The AAC3 gene is predominantly expressed under anaerobic growth conditions (Gawaz et al., 1990;Drgon et al., 1991). The Sa1l protein represents another member of the mitochondrial AAC family, which also catalyzes ADP/ATP exchange, but it differs from other AAC family members in that it contains an extended N-terminal hydrophilic region proposed to be involved in Ca 2ϩ binding (Chen, 2004). Deletion of both the AAC2 and AAC3 genes, or of the AAC2 and SAL1 genes, results in a synthetic lethal phenotype under anaerobic and aerobic conditions, respectively (Drgon et al., 1992;Chen, 2004). Thus in addition to their role in ADP/ATP exchange, these AAC family members perform additional overlapping function(s) that are essential for cell viability. It is currently unclear, however, what essential role(s) these AAC family members may have.The oxidativ...

The yeast Oxa1 protein is involved in the biogenesis of the mitochondrial oxidative phosphorylation (OXPHOS) machinery. The involvement of Oxa1 in the assembly of the cytochrome oxidase (COX) complex, where it facilitates the cotranslational membrane insertion of mitochondrially encoded COX subunits, is well documented. In this study we have addressed the role of Oxa1, and its sequence-related protein Cox18/Oxa2, in the biogenesis of the F 1 F o -ATP synthase complex. We demonstrate that Oxa1, but not Cox18/Oxa2, directly supports the assembly of the membrane embedded F o -sector of the ATP synthase. Oxa1 was found to physically interact with newly synthesized mitochondrially encoded Atp9 protein in a posttranslational manner and in a manner that is not dependent on the C-terminal, matrix-localized region of Oxa1. The stable manner of the Atp9-Oxa1 interaction is in contrast to the cotranslational and transient interaction previously observed for the mitochondrially encoded COX subunits with Oxa1. In the absence of Oxa1, Atp9 was observed to assemble into an oligomeric complex containing F 1 -subunits, but its further assembly with subunit 6 (Atp6) of the F o -sector was perturbed. We propose that by directly interacting with newly synthesized Atp9 in a posttranslational manner, Oxa1 is required to maintain the assembly competence of the Atp9-F 1 -subcomplex for its association with Atp6.

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.

The enzyme complexes involved in mitochondrial oxidative phosphorylation are organized into higher ordered assemblies termed supercomplexes. Subunits e and g (Su e and Su g, respectively) are catalytically nonessential subunits of the F 1 F 0 -ATP synthase whose presence is required to directly support the stable dimerization of the ATP synthase complex. We report here that Su g and Su e are also important for securing the correct organizational state of the cytochrome bc 1 -cytochrome oxidase (COX) supercomplex. Mitochondria isolated from the ⌬su e and ⌬su g null mutant strains exhibit decreased levels of COX enzyme activity but appear to have normal COX subunit protein levels. An altered stoichiometry of the cytochrome bc 1 -COX supercomplex was observed in mitochondria deficient in Su e and/or Su g, and a perturbation in the association of Cox4, a catalytically important subunit of the COX complex, was also detected. In addition, an increase in the level of the TIM23 translocase associated with the cytochrome bc 1 -COX supercomplex is observed in the absence of Su e and Su g. Together, our data highlight that a further level of complexity exists between the oxidative phosphorylation supercomplexes, whereby the organizational state of one complex, i.e. the ATP synthase, may influence that of another supercomplex, namely the cytochrome bc 1 -COX complex.