Rosemary A. Stuart

Yeast mitochondrial F1F0‐ATP synthase exists as a dimer: identification of three dimer‐specific subunits

Using the technique of blue native gel electrophoresis, the oligomeric state of the yeast mitochondrial F1F0‐ATP synthase was analysed. Solubilization of mitochondrial membranes with low detergent to protein ratios led to the identification of the dimeric state of the ATP synthase. Analysis of the subunit composition of the dimer, in comparison with the monomer, revealed the presence of three additional small proteins. These dimer‐specific subunits of the ATP synthase were identified as the recently described subunit e/Tim11 (Su e/Tim11), the putative subunit g homolog (Su g) and a new component termed subunit k (Su k). Although, as shown here, these three proteins are not required for the formation of enzymatically active ATP synthase, Su e/Tim11 and Su g are essential for the formation of the dimeric state. Su e/Tim11 appears to play a central role in this dimerization process. The dimer‐specific subunits are associated with the membrane bound F0‐sector. The F0‐sector may thereby be involved in the dimerization of two monomeric F1F0‐ATP synthase complexes. We speculate that the F1F0‐ATP synthase of yeast, like the other complexes of oxidative phosphorylation, form supracomplexes to optimize transduction of energy and to enhance the stability of the complex in the membrane.

Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5.

Import of nuclear-encoded precursor proteins into mitochondria and their subsequent sorting into mitochondrial subcompartments is mediated by translocase enzymes in the mitochondrial outer and inner membranes. Precursor proteins carrying amino-terminal targeting signals are translocated into the matrix by the integral inner membrane proteins Tim23 and Tim17 in cooperation with Tim44 and mitochondrial Hsp70 (refs 4-7). We describe here the discovery of a new pathway for the transport of members of the mitochondrial carrier family and other inner membrane proteins that contain internal targeting signals. Two related proteins in the intermembrane space, Tim10/Mrs11 (ref. 8) and Tim12/Mrs5 (ref. 9), interact sequentially with these precursors and facilitate their translocation across the outer membrane, irrespective of the membrane potential. Tim10 and Tim12 are found in a complex with Tim22, which takes over the precursor and mediates its membrane-potential-dependent insertion into the inner membrane. This interaction of Tim10 and Tim12 with the precursors depends on the presence of divalent metal ions. Both proteins contain a zinc-finger-like motif with four cysteines and bind equimolar amounts of zinc ions.

Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA.

Oxa1p is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1p has been shown previously to directly facilitate the export of the N‐terminal domains of membrane proteins across the inner membrane to the intermembrane space of mitochondria. Here we report on a general role of Oxa1p in the membrane insertion of proteins. (i) The function of Oxa1p is not limited to the insertion of membrane proteins that undergo N‐terminal tail export; rather, it also extends to the insertion of other polytopic proteins such as the mitochondrially encoded Cox1p and Cox3p proteins. These are proteins whose N‐termini are retained in the mitochondrial matrix. (ii) Oxa1p interacts directly with these substrates prior to completion of their synthesis. (iii) The interaction of Oxa1p with its substrates is particularly strong when nascent polypeptide chains are inserted into the inner membrane, suggesting a direct function of Oxa1p in co‐translational insertion from the matrix. Taken together, we conclude that the Oxa1 complex represents a general membrane protein insertion machinery in the inner membrane of mitochondria.

AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria.

The mechanism of selective protein degradation of membrane proteins in mitochondria has been studied employing a model protein that is subject to rapid proteolysis within the inner membrane. Protein degradation was mediated by two different proteases: (i) the m‐AAA protease, a protease complex consisting of multiple copies of the ATP‐dependent metallopeptidases Yta1Op (Afg3p) and Yta12p (Rcalp); and (ii) by Ymelp (Ytallp) that also is embedded in the inner membrane. Ymelp, highly homologous to Yta1Op and Yta12p, forms a complex of approximately 850 kDa in the inner membrane and exerts ATP‐dependent metallopeptidase activity. While the m‐AAA protease exposes catalytic sites to the mitochondrial matrix, Ymelp is active in the intermembrane space. The Ymelp complex was therefore termed ‘i‐AAA protease’. Analysis of the proteolytic fragments indicated cleavage of the model polypeptide at the inner and outer membrane surface and within the membrane‐spanning domain. Thus, two AAA proteases with their catalytic sites on opposite membrane surfaces constitute a novel proteolytic system for the degradation of membrane proteins in mitochondria.

Yeast Oxa1 interacts with mitochondrial ribosomes: the importance of the C-terminal region of Oxa1

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.

Internal targeting signal of the BCS1 protein: a novel mechanism of import into mitochondria.

The BCS1 protein is anchored in the mitochondrial inner membrane via a single transmembrane domain and has an N(out)‐C(in) topology. Unlike the majority of nuclear encoded mitochondrial preproteins, the BCS1 protein does not contain an N‐terminal targeting sequence. A positively charged segment of amino acids which is located immediately C‐terminal to the transmembrane domain acts as an internal targeting signal. In order to function, we postulate that this sequence co‐operates with the transmembrane domain to form a tight hairpin loop structure. This loop is translocated across the inner membrane via the MIM/mt‐Hsp70 machinery in a membrane potential‐dependent manner. This novel mechanism of import and sorting of the BCS1 protein is proposed to represent a more general mechanism used by a number of inner membrane proteins.

Oxa1p mediates the export of the N‐ and C‐termini of pCoxII from the mitochondrial matrix to the intermembrane space

Oxa1p is a mitochondrial protein reported to be involved in the assembly of the cytochrome oxidase complex. In the absence of a functional Oxa1p, subunit II of the cytochrome oxidase accumulates as its precursor form (pCoxII). Using mitochondria isolated from a yeast strain bearing a temperature sensitive mutation in the Oxa1p, pet ts1402, we have analyzed the function of the Oxa1p protein. We demonstrate that the accumulation of pCoxII in the pet ts1402 mitochondria does not reflect a compromised Imp1p activity in this mutant. Furthermore, measurement of the membrane potential has shown it to be sufficient to support the export of CoxII from the matrix. Rather, we found that newly synthesized pCoxII accumulates in the matrix of the pet ts1402 mitochondria, because export across the inner membrane is inhibited in the pet ts1402 mitochondria. In conclusion, Oxa1p mediates the export of the N‐ and C‐termini of the mitochondrially encoded subunit II of cytochrome oxidase from the matrix to the intermembrane space.

Mitochondrial molecular chaperones: their role in protein translocation

After synthesis in the cytosol, most mitochondrial proteins must traverse mitochondrial membranes to reach their functional location. During this process, proteins become unfolded and then refold to attain their native conformation after crossing the lipid bilayers. Mitochondrial molecular chaperones play an essential mechanistic role at various steps of this process. They facilitate presequence translocation, unfolding of the cytosol-localized domains of precursor proteins, movement across the mitochondrial membranes and, finally, folding of newly imported proteins within the matrix.

Insertion into the mitochondrial inner membrane of a polytopic protein, the nuclear-encoded Oxa1p.

Oxa1p, a nuclear‐encoded protein of the mitochondrial inner membrane with five predicted transmembrane (TM) segments is synthesized as a precursor (pOxa1p) with an N‐terminal presequence. It becomes imported in a process requiring the membrane potential, matrix ATP, mt‐Hsp70 and the mitochondrial processing peptidase (MPP). After processing, the negatively charged N‐terminus of Oxa1p (∼90 amino acid residues) is translocated back across the inner membrane into the intermembrane space and thereby attains its native Nout–Cin orientation. This export event is dependent on the membrane potential. Chimeric preproteins containing N‐terminal stretches of increasing lengths of Oxa1p fused on mouse dehydrofolate reductase (DHFR) were imported into isolated mitochondria. In each case, their DHFR moieties crossed the inner membrane into the matrix. Thus Oxa1p apparently does not contain a stop transfer signal. Instead the TM segments are inserted into the membrane from the matrix side in a pairwise fashion. The sorting pathway of pOxa1p is suggested to combine the pathways of general import into the matrix with a bacterial‐type export process. We postulate that at least two different sorting pathways exist in mitochondria for polytopic inner membrane proteins, the evolutionarily novel pathway for members of the ADP/ATP carrier family and a conserved Oxa1p‐type pathway.

Insertion of proteins into the inner membrane of mitochondria: the role of the Oxa1 complex

The inner mitochondrial membrane harbors a large number of proteins that display a wide range of topological arrangements. The majority of these proteins are encoded in the cells nucleus, but a few polytopic proteins, all subunits of respiratory chain complexes are encoded by the mitochondrial genome. A number of distinct sorting mechanisms exist to direct these proteins into the mitochondrial inner membrane. One of these pathways involves the export of proteins from the matrix into the inner membrane and is used by both proteins synthesized within the mitochondria, as well as by a subset of nuclear encoded proteins. Prior to embarking on the export pathway, nuclear encoded proteins using this sorting route are initially imported into the mitochondrial matrix from the cytosol, their site of synthesis. Protein export from the matrix into the inner membrane bears similarities to Sec-independent protein export in bacteria and requires the function of the Oxa1 protein. Oxa1 is a component of a general protein insertion site in yeast mitochondrial inner membrane used by both nuclear and mitochondrial DNA encoded proteins. Oxa1 is a member of the conserved Oxa1/YidC/Alb3 protein family found throughout prokaryotes throughout eukaryotes (where it is found in mitochondria and chloroplasts). The evidence to demonstrate that the Oxa1/YidC/Alb3 protein family represents a novel evolutionarily conserved membrane insertion machinery is reviewed here.