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.     

Translocation of mitochondrially synthesized Cox2 domains from the matrix to the intermembrane space

The N-terminal and C-terminal domains of mitochondrially synthesized cytochrome c oxidase subunit II, Cox2, are translocated through the inner membrane to the intermembrane space (IMS). We investigated the distinct mechanisms of N-tail and C-tail export by analysis of epitope-tagged Cox2 variants encoded in Saccharomyces cerevisiae mitochondrial DNA. Both the N and C termini of a truncated protein lacking the Cox2 C-terminal domain were translocated to the IMS via a pathway dependent upon the conserved translocase Oxa1. The topology of this Cox2 variant, accumulated at steady state, was largely but not completely unaffected in mutants lacking proteins required for export of the C-tail domain, Cox18 and Mss2. C-tail export was blocked by truncation of the last 40 residues from the C-tail domain, indicating that sequence and/or structural features of this domain are required for its translocation. Mss2, a peripheral protein bound to the inner surface of the inner membrane, coimmunoprecipitated with full-length newly synthesized Cox2, whose leader peptide had already been cleaved in the IMS. Our data suggest that the C-tail domain is recognized posttranslationally by a specialized translocation apparatus after the N-tail has been translocated by Oxa1.     

A yeast mitochondrial membrane methyltransferase-like protein can compensate for oxa1 mutations

Members of the Oxa1p/Alb3/YidC family mediate the insertion of various organelle or bacterial hydrophobic proteins into membranes. They present at least five transmembrane segments (TM) linked by hydrophilic domains located on both sides of the membrane. To examine how Oxa1p structure relates to its function, we have introduced point mutations and large deletions into various domains of the yeast mitochondrial protein. These mutants allowed us to show the importance of the first TM domain as well as a synergistic interaction between the first loop and the C-terminal tail, which both protrude into the matrix. These mutants also led to the isolation of a high copy suppressor, OMS1, which encodes a member of the methyltransferase family. Overexpression of OMS1 seems to increase the steady-state level of both the mutant and wild-type Oxa1p. We show that Oms1p is a mitochondrial inner membrane protein inserted independently of Oxa1p. Oms1p presents one TM and a N-in C-out topology with the C-terminal domain carrying the methyltransferase-like domain. A conserved motif within this domain is essential for the suppression of oxa1 mutations. We discuss the possible role of Oms1p on Oxa1p intermembrane space domain.     

Roles of Oxa1-related inner-membrane translocases in assembly of respiratory chain complexes

Members of the family of the polytopic inner membrane proteins are related to Saccharomyces cerevisiae Oxa1 function in the assembly of energy transducing complexes of mitochondria and chloroplasts. Here we focus on the two mitochondrial members of this family, Oxa1 and Cox18, reviewing studies on their biogenesis as well as their functions, reflected in the phenotypic consequences of their absence in various organisms. In yeast, cytochrome c oxidase subunit II (Cox2) is a key substrate of these proteins. Oxa1 is required for co-translational translocation and insertion of Cox2, while Cox18 is necessary for the export of its C-terminal domain. Genetic and biochemical strategies have been used to investigate the functions of distinct domains of Oxa1 and to identify its partners in protein insertion/translocation. Recent work on the related bacterial protein YidC strongly indicates that it is capable of functioning alone as a translocase for hydrophilic domains and an insertase for TM domains. Thus, the Oxa1 and Cox18 probably catalyze these reactions directly in a co- and/or posttranslational way. In various species, Oxa1 appears to assist in the assembly of different substrate proteins, although it is still unclear how Oxa1 recognizes its substrates, and whether additional factors participate in this beyond its direct interaction with mitochondrial ribosomes, demonstrated in S. cerevisiae. Oxa1 is capable of assisting posttranslational insertion and translocation in isolated mitochondria, and Cox18 may posttranslationally translocate its only known substrate, the Cox2 C-terminal domain, in vivo. Detailed understanding of the mechanisms of action of these two proteins must await the resolution of their structure in the membrane and the development of a true in vitro mitochondrial translation system.     

Multiple roles of the Cox20 chaperone in assembly of Saccharomyces cerevisiae cytochrome c oxidase

The Cox2 subunit of Saccharomyces cerevisiae cytochrome c oxidase is synthesized in the mitochondrial matrix as a precursor whose leader peptide is rapidly processed by the inner membrane protease following translocation to the intermembrane space. Processing is chaperoned by Cox20, an integral inner membrane protein whose hydrophilic domains are located in the intermembrane space, and Cox20 remains associated with mature, unassembled Cox2. The Cox2 C-tail domain is exported post-translationally by the highly conserved translocase Cox18 and associated proteins. We have found that Cox20 is required for efficient export of the Cox2 C-tail. Furthermore, Cox20 interacts by co-immune precipitation with Cox18, and this interaction requires the presence of Cox2. We therefore propose that Cox20 binding to Cox2 on the trans side of the inner membrane accelerates dissociation of newly exported Cox2 from the Cox18 translocase, promoting efficient cycling of the translocase. The requirement for Cox20 in cytochrome c oxidase assembly and respiratory growth is partially bypassed by yme1, mgr1 or mgr3 mutations, each of which reduce i-AAA protease activity in the intermembrane space. Thus, Cox20 also appears to stabilize unassembled Cox2 against degradation by the i-AAA protease. Pre-Cox2 leader peptide processing by Imp1 occurs in the absence of Cox20 and i-AAA protease activity, but is greatly reduced in efficiency. Under these conditions some mature Cox2 is assembled into cytochrome c oxidase allowing weak respiratory growth. Thus, the Cox20 chaperone has important roles in leader peptide processing, C-tail export, and stabilization of Cox2.     

Absence of the mitochondrial AAA protease Yme1p restores F0-ATPase subunit accumulation in an oxa1 deletion mutant of Saccharomyces cerevisiae

The nuclear gene OXA1 encodes a protein located within the mitochondrial inner membrane that is required for the biogenesis of both cytochrome c oxidase (Cox) and ATPase. In the absence of Oxa1p, the translocation of the mitochondrially encoded subunit Cox2p to the intermembrane space (also referred to as export) is prevented, and it has been proposed that Oxa1p could be a component of a general mitochondrial export machinery. We have examined the role of Oxa1p in light of its relationships with two mitochondrial proteases, the matrix protease Afg3p-Rca1p and the intermembrane space protease Yme1p, by analyzing the assembly and activity of the Cox and ATPase complexes in Deltaoxa1, Deltaoxa1Deltaafg3, and Deltaoxa1Deltayme1 mutants. We show that membrane subunits of both complexes are specifically degraded in the absence of Oxa1p. Neither Afg3p nor Yme1p is responsible for the degradation of Cox subunits. However, the F(0) subunits Atp4p, Atp6p, and Atp17p are stabilized in the Deltaoxa1Deltayme1 double mutant, and oligomycin-sensitive ATPase activity is restored, showing that the increased stability of the ATPase subunits allows significant translocation and assembly to occur even in the absence of Oxa1p. These results suggest that Oxa1p is not essential for the export of ATPase subunits. In addition, although respiratory function is dispensable in Saccharomyces cerevisiae, we show that the simultaneous inactivation of AFG3 and YME1 is lethal and that the essential function does not reside in their protease activity.     

Transport of proteins to the mitochondrial intermembrane space: the ''sorting'' domain of the cytochrome c1 presequence is a stop-transfer sequence specific for the mitochondrial inner membrane.

The presequence of yeast cytochrome c1 (an inner membrane protein protruding into the intermembrane space) contains a matrix-targeting domain and an intramitochondrial sorting domain. This presequence transports attached subunit IV of cytochrome c oxidase into the intermembrane space (van Loon et al. (1987) EMBO J., 6, 2433-2439). In order to determine how this fusion protein reaches the intermembrane space, we studied the kinetics of its import into isolated mitochondria or mitoplasts and its accumulation in the various submitochondrial compartments. The imported, uncleaved fusion precursor and a cleavage intermediate were bound to the inner membrane and were always exposed to the intermembrane space; they were never found at the matrix side of the inner membrane. In contrast, analogous import experiments with the authentic subunit IV precursor, or the precursor of the iron-sulphur protein of the cytochrome bc1 complex also an inner membrane protein exposed to the intermembrane space), readily showed that these precursors were initially transported across both mitochondrial membranes. We conclude that the intramitochondrial sorting domain within the cytochrome c1 presequence prevents transport of attached proteins across the inner, but not the outer membrane: it is a stop-transfer sequence for the inner membrane. Since the presequence of the iron-sulphur protein lacks such 'stop-transfer' domain, it acts by a different mechanism.     

Mitochondria use different mechanisms for transport of multispanning membrane proteins through the intermembrane space

The mitochondrial inner membrane contains numerous multispanning integral proteins. The precursors of these hydrophobic proteins are synthesized in the cytosol and therefore have to cross the mitochondrial outer membrane and intermembrane space to reach the inner membrane. While the import pathways of noncleavable multispanning proteins, such as the metabolite carriers, have been characterized in detail by the generation of translocation intermediates, little is known about the mechanism by which cleavable preproteins of multispanning proteins, such as Oxa1, are transferred from the outer membrane to the inner membrane. We have identified a translocation intermediate of the Oxa1 preprotein in the translocase of the outer membrane (TOM) and found that there are differences from the import mechanisms of carrier proteins. The intermembrane space domain of the receptor Tom22 supports the stabilization of the Oxa1 intermediate. Transfer of the Oxa1 preprotein to the inner membrane is not affected by inactivation of the soluble TIM complexes. Both the inner membrane potential and matrix heat shock protein 70 are essential to release the preprotein from the TOM complex, suggesting a close functional cooperation of the TOM complex and the presequence translocase of the inner membrane. We conclude that mitochondria employ different mechanisms for translocation of multispanning proteins across the aqueous intermembrane space.     

Saccharomyces cerevisiae Cox18 complements the essential Sec-independent function of Escherichia coli YidC

Members of the YidC/Oxa1/Alb3 protein family function in the biogenesis of membrane proteins in bacteria, mitochondria and chloroplasts. In Escherichia coli, YidC plays a key role in the integration and assembly of many inner membrane proteins. Interestingly, YidC functions both in concert with the Sec-translocon and as a separate insertase independent of the translocon. Mitochondria of higher eukaryotes contain two distant homologues of YidC: Oxa1 and Cox18/Oxa2. Oxa1 is required for the insertion of membrane proteins into the mitochondrial inner membrane. Cox18/Oxa2 plays a poorly defined role in the biogenesis of the cytochrome c oxidase complex. Employing a genetic complementation approach by expressing the conserved region of yeast Cox18 in E. coli, we show here that Cox18 is able to complement the essential Sec-independent function of YidC. This identifies Cox18 as a bona fide member of the YidC/Oxa1/Alb3 family.     

Rmd9p controls the processing/stability of mitochondrial mRNAs and its overexpression compensates for a partial deficiency of oxa1p in Saccharomyces cerevisiae

Oxa1p is a key component of the general membrane insertion machinery of eukaryotic respiratory complex subunits encoded by the mitochondrial genome. In this study, we have generated a respiratory-deficient mutant, oxa1-E65G-F229S, that contains two substitutions in the predicted intermembrane space domain of Oxa1p. The respiratory deficiency due to this mutation is compensated for by overexpressing RMD9. We show that Rmd9p is an extrinsic membrane protein facing the matrix side of the mitochondrial inner membrane. Its deletion leads to a pleiotropic effect on respiratory complex biogenesis. The steady-state level of all the mitochondrial mRNAs encoding respiratory complex subunits is strongly reduced in the Deltarmd9 mutant, and there is a slight decrease in the accumulation of two RNAs encoding components of the small subunit of the mitochondrial ribosome. Overexpressing RMD9 leads to an increase in the steady-state level of mitochondrial RNAs, and we discuss how this increase could suppress the oxa1 mutations and compensate for the membrane insertion defect of the subunits encoded by these mRNAs.     

Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p.

To study in vivo the export of mitochondrially synthesized protein from the matrix to the intermembrane space, we have fused a synthetic mitochondrial gene, ARG8m, to the Saccharomyces cerevisiae COX2 gene in mitochondrial DNA. The Arg8mp moiety was translocated through the inner membrane when fused to the Cox2p C terminus by a mechanism dependent on topogenic information at least partially contained within the exported Cox2p C-terminal tail. The pre-Cox2p leader peptide did not signal translocation. Export of the Cox2p C-terminal tail, but not the N-terminal tail, was dependent on the inner membrane potential. The mitochondrial export system does not closely resemble the bacterial Sec translocase. However, normal translocation of both exported domains of Cox2p was defective in cells lacking the widely conserved inner membrane protein Oxa1p.     

Evolution of mitochondrial oxa proteins from bacterial YidC. Inherited and acquired functions of a conserved protein insertion machinery

Members of the Oxa1/YidC family are involved in the biogenesis of membrane proteins. In bacteria, YidC catalyzes the insertion and assembly of proteins of the inner membrane. Mitochondria of animals, fungi, and plants harbor two distant homologues of YidC, Oxa1 and Cox18/Oxa2. Oxa1 plays a pivotal role in the integration of mitochondrial translation products into the inner membrane of mitochondria. It contains a C-terminal ribosome-binding domain that physically interacts with mitochondrial ribosomes to facilitate the co-translational insertion of nascent membrane proteins. The molecular function of Cox18/Oxa2 is not well understood. Employing a functional complementation approach with mitochondria-targeted versions of YidC we show that YidC is able to functionally replace both Oxa1 and Cox18/Oxa2. However, to integrate mitochondrial translation products into the inner membrane of mitochondria, the ribosome-binding domain of Oxa1 has to be appended onto YidC. On the contrary, the fusion of the ribosome-binding domain onto YidC prevents its ability to complement COX18 mutants suggesting an indispensable post-translational activity of Cox18/Oxa2. Our observations suggest that during evolution of mitochondria from their bacterial ancestors the two descendents of YidC functionally segregated to perform two distinct activities, one co-translational and one post-translational.     

Insertion of mitochondrial DNA-encoded F1F0-ATPase subunit 8 across the mitochondrial inner membrane in vitro

Cytochrome oxidase subunits I, II, and III, the mitochondrial DNA-encoded proteins, are inserted across the inner membrane by the Oxa1p-containing translocator in a membrane potential-dependent manner. Oxa1p is also involved in the insertion of the cytoplasmically synthesized precursor of Oxa1p itself into the inner membrane from the matrix via the conservative sorting pathway. The mechanism of insertion of the other mitochondrially synthesized proteins, however, is unexplored. The insertion of the mitochondrial DNA-encoded subunit 8 of F(1)F(0)-ATPase (Su8) across the inner membrane was analyzed in vitro using the inverted inner membrane vesicles and the Escherichia coli lysate-synthesized substrate. This assay revealed that the N-terminal segment of Su8 inserted across the membrane to the intermembrane space and assumed the correct trans-cis topology depending on the mitochondrial matrix fraction. This translocation reaction was similar to those of Sec-independent, direct insertion pathways of E. coli and chloroplast thylakoid membranes. (i) It required neither nucleotide triphosphates nor membrane potential, and hydrophobic forces drove the process. (ii) It did not require protease-sensitive membrane components facing the matrix space. (iii) It could be inserted across liposomes in the correct topology in a matrix fraction-dependent manner. Thus, a novel mechanism conserved in bacteria and chloroplasts also functions in the insertion of Su8 across the mitochondrial inner membrane.     

Ribosome binding to the Oxa1 complex facilitates co-translational protein insertion in mitochondria

The Oxa1 translocase of the mitochondrial inner membrane facilitates the insertion of both mitochondrially and nuclear-encoded proteins from the matrix into the inner membrane. Most mitochondrially encoded proteins are hydrophobic membrane proteins which are integrated into the lipid bilayer during their synthesis on mitochondrial ribosomes. The molecular mechanism of this co-translational insertion process is unknown. Here we show that the matrix-exposed C-terminus of Oxa1 forms an alpha-helical domain that has the ability to bind to mitochondrial ribosomes. Deletion of this Oxa1 domain strongly diminished the efficiency of membrane insertion of subunit 2 of cytochrome oxidase, a mitochondrially encoded substrate of the Oxa1 translocase. This suggests that co-translational membrane insertion of mitochondrial translation products is facilitated by a physical interaction of translation complexes with the membrane-bound translocase.     

Mammalian Oxa1 protein is useful for assessment of submitochondrial protein localization and mitochondrial membrane integrity

Taking advantage of the unique topology of oxidase assembly 1 (Oxa1) protein, a mitochondrial inner membrane protein with N (intermembrane space)-C (matrix) orientation, we explored the usefulness of the protein as a marker for submitochondrial protein localization. Mammalian Oxa1 protein exhibited different proteolytic patterns depending on mitochondrial membrane integrity, and in mitochondria with a disrupted outer membrane and outer and inner membranes, the proteolytic patterns of Oxa1 protein were consistent with those of mitochondrial intermembrane space and matrix marker proteins, respectively, suggesting that Oxa1 protein, a single molecule, can serve as a versatile submitochondrial localization marker that doubles as a membrane integrity marker.     

The Membrane Insertase Oxa1 Is Required for Efficient Import of Carrier Proteins into Mitochondria

Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady‐state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.     

Cox17 is functional when tethered to the mitochondrial inner membrane

Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion. Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex. To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2. The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17. The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion. The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Delta cells. These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space. Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding. The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein. This motif can be spatially separated from the N-terminal copper-binding functional motif. Possible roles of the C-terminal motif are discussed.     

N-terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic "positive-inside" rule of membrane protein topology.

Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N- and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for sec-independent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.     

Mba1, a novel component of the mitochondrial protein export machinery of the yeast Saccharomyces cerevisiae

The biogenesis of mitochondria requires the integration of many proteins into the inner membrane from the matrix side. The inner membrane protein Oxa1 plays an important role in this process. We identified Mba1 as a second mitochondrial component that is required for efficient protein insertion. Like Oxa1, Mba1 specifically interacts both with mitochondrial translation products and with conservatively sorted, nuclear-encoded proteins during their integration into the inner membrane. Oxa1 and Mba1 overlap in function and substrate specificity, but both can act independently of each other. We conclude that Mba1 is part of the mitochondrial protein export machinery and represents the first component of a novel Oxa1-independent insertion pathway into the mitochondrial inner membrane.     

The Mitochondrial Oxidase Assembly Protein1 (Oxa1) Insertase Forms a Membrane Pore in Lipid Bilayers

The inner membrane of mitochondria is especially protein-rich. To direct proteins into the inner membrane, translocases mediate transport and membrane insertion of precursor proteins. Although the majority of mitochondrial proteins are imported from the cytoplasm, core subunits of respiratory chain complexes are inserted into the inner membrane from the matrix. Oxa1, a conserved membrane protein, mediates the insertion of mitochondrion-encoded precursors into the inner mitochondrial membrane. The molecular mechanism by which Oxa1 mediates insertion of membrane spans, entailing the translocation of hydrophilic domains across the inner membrane, is still unknown. We investigated if Oxa1 could act as a protein-conducting channel for precursor transport. Using a biophysical approach, we show that Oxa1 can form a pore capable of accommodating a translocating protein segment. After purification and reconstitution, Oxa1 acts as a cation-selective channel that specifically responds to mitochondrial export signals. The aqueous pore formed by Oxa1 displays highly dynamic characteristics with a restriction zone diameter between 0.6 and 2 nm, which would suffice for polypeptide translocation across the membrane. Single channel analyses revealed four discrete channels per active unit, suggesting that the Oxa1 complex forms several cooperative hydrophilic pores in the inner membrane. Hence, Oxa1 behaves as a pore-forming translocase that is regulated in a membrane potential and substrate-dependent manner.