Identification of Protein-Protein and Protein-Ribosome Interacting Regions of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L

The mammalian mitochondrial inner membrane protein Oxa1L is involved in the insertion of a number of mitochondrial translation products into the inner membrane. During this process, the C-terminal tail of Oxa1L (Oxa1L-CTT) binds mitochondrial ribosomes and is believed to coordinate the synthesis and membrane insertion of the nascent chains into the membrane. The C-terminal tail of Oxa1L does not contain any Cys residues. Four variants of this protein with a specifically placed Cys residue at position 4, 39, 67, or 94 of Oxa1L-CTT have been prepared. These Cys residues have been derivatized with a fluorescent probe, tetramethylrhodamine-5-maleimide, for biophysical studies. Oxa1L-CTT forms oligomers cooperatively with a binding constant in the submicromolar range. Fluorescence anisotropy and fluorescence lifetime measurements indicate that contacts near a long helix close to position 39 of Oxa1L-CTT occur during oligomer formation. Fluorescence correlation spectroscopy measurements demonstrate that all of the Oxa1L-CTT derivatives bind to mammalian mitochondrial ribosomes. Steady-state fluorescence quenching and fluorescence lifetime data indicate that there are extensive contacts between Oxa1L-CTT and the ribosome-encompassing regions around positions 39, 67, and 94. The results of this study suggest that Oxa1L-CTT undergoes conformational changes and induced oligomer formation when it binds to the ribosome.     

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.     

Mapping of the Saccharomyces cerevisiae Oxa1-Mitochondrial Ribosome Interface and Identification of MrpL40, a Ribosomal Protein in Close Proximity to Oxa1 and Critical for Oxidative Phosphorylation Complex Assembly

The Oxa1 protein plays a central role in facilitating the cotranslational insertion of the nascent polypeptide chains into the mitochondrial inner membrane. Mitochondrially encoded proteins are synthesized on matrix-localized ribosomes which are tethered to the inner membrane and in physical association with the Oxa1 protein. In the present study we used a chemical cross-linking approach to map the Saccharomyces cerevisiae Oxa1-ribosome interface, and we demonstrate here a close association of Oxa1 and the large ribosomal subunit protein, MrpL40. Evidence to indicate that a close physical and functional relationship exists between MrpL40 and another large ribosomal protein, the Mrp20/L23 protein, is also provided. MrpL40 shares sequence features with the bacterial ribosomal protein L24, which like Mrp20/L23 is known to be located adjacent to the ribosomal polypeptide exit site. We propose therefore that MrpL40 represents the Saccharomyces cerevisiae L24 homolog. MrpL40, like many mitochondrial ribosomal proteins, contains a C-terminal extension region that bears no similarity to the bacterial counterpart. We show that this C-terminal mitochondria-specific region is important for MrpL40''s ability to support the synthesis of the correct complement of mitochondrially encoded proteins and their subsequent assembly into oxidative phosphorylation complexes.The mitochondrial genome encodes a small, but important, number of proteins (8). These proteins are predominantly essential components of the mitochondrial oxidative phosphorylation (OXPHOS) machinery. In the yeast Saccharomyces cerevisiae the proteins encoded by the mitochondrial DNA (mtDNA) include cytochrome c oxidase subunits Cox1, Cox2, and Cox3, cytochrome b of the cytochrome bc₁ complex, F₁F_o-ATP synthase subunits Atp6, Atp8, and Atp9, and the small ribosomal subunit component Var1. With the exception of Var1, these mitochondrially encoded proteins are integral membrane proteins which become inserted into the inner membrane during their synthesis on mitochondrial ribosomes tethered to the inner membrane (11, 19, 29, 32, 34). The cotranslational membrane insertion of these proteins is achieved by maintaining a close physical association of the ribosomes to the inner membrane at sites where the insertion machinery exists (19, 31, 32).Oxa1 is an inner membrane protein that forms a central component of the insertion machinery, whose presence is required for the cotranslational membrane insertion of the mitochondrially encoded proteins (4-6, 15-17). The Oxa1 protein has been shown to physically associate with the ribosomes and more specifically with the large ribosomal subunit. Matrix-exposed elements of the Oxa1 protein, such as its hydrophilic C-terminal tail, support this Oxa1-ribosome interaction (19, 32). Furthermore, in intact mitochondria we have previously demonstrated that Oxa1 can be chemically cross-linked to Mrp20, a component of the large ribosomal subunit (19). Mrp20 is homologous to the bacterial ribosomal protein L23, a component known from the structural analysis of the ribosomes to be located next to the polypeptide exit site of the large ribosomal subunit (3, 10, 23, 27, 30). Thus, it was concluded that Oxa1, the site of membrane insertion into the inner membrane, exists in close physical proximity to the large ribosomal subunit and specifically to that region of the ribosomes where the nascent chain emerges. This close physical relationship between ribosomal components and the Oxa1 insertion site has been proposed to support a tight coordination between the protein translation and membrane insertion events (19, 31, 32). Given the strong hydrophobicity of the OXPHOS complex subunits which are encoded by the mitochondrial DNA and synthesized by these ribosomes, a close coupling of the translation and insertion events is proposed to ensure that the hydrophobic nascent chains are directly inserted into the membrane during their synthesis. The exposure of hydrophobic nascent chains to the hydrophilic matrix space may promote their aggregation and thus incompetency for subsequence membrane insertion.In bacteria, the L23 protein has been implicated to play a direct role in the cotranslational insertion of proteins into the membrane (7, 13, 24, 33). Thus, it is possible that proteins adjacent to the polypeptide exit site of mitochondrial ribosomes may be directly involved in targeting ribosomes to specific regions of the inner membrane where the membrane insertion and subsequent assembly events occur. The mitochondrial ribosomes resemble their prokaryotic ancestors in some respects, e.g., antibiotic sensitivity, but they differ in a number of important ways (1, 12, 22, 30). In general, the protein content of the mitochondrial ribosomes is greater than their bacterial counterparts. This increase in protein content is largely attributed to the fact that the mitochondrial ribosomal proteins are larger in size than their bacterial homologs. Over the course of evolution, many of the mitochondrial ribosomal proteins have acquired novel extensions, new domains, in addition to their bacterial homology domains. These acquired extensions not only include N-terminal (often cleavable) signals to target these proteins (nuclear encoded) to the mitochondria but also in many instances large C-terminal extensions, which are unique to the mitochondrial ribosomal proteins and have thus been termed “mitospecific domains” (12, 30). Largely uncharacterized, the functional relevance of these various mitospecific domains of the ribosomal proteins remains unknown. It is speculated that some (or all) of these mitospecific domains serve to ensure that the ribosome becomes assembled and is translationally active while bound to the inner membrane surface.In the present study we sought to further characterize the interaction of the mitochondrial ribosome with the Oxa1 protein. We show here that MrpL40, a large ribosomal subunit component, is physically close to both the Mrp20 and Oxa1 proteins, demonstrating the proximity of MrpL40 to both the ribosomal polypeptide exit site and the Oxa1 membrane insertion site. MrpL40 contains a large C-terminal mitospecific domain, which includes a predicted α-helical region at its extreme C-terminal end. The results presented here highlight that the integrity of this domain of MrpL40 is crucial to ensure ribosome translational fidelity and subsequent OXPHOS complex assembly.     

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.     

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.     

The ER membrane complex (EMC) can functionally replace the Oxa1 insertase in mitochondria

Two multisubunit protein complexes for membrane protein insertion were recently identified in the endoplasmic reticulum (ER): the guided entry of tail anchor proteins (GET) complex and ER membrane complex (EMC). The structures of both of their hydrophobic core subunits, which are required for the insertion reaction, revealed an overall similarity to the YidC/Oxa1/Alb3 family members found in bacteria, mitochondria, and chloroplasts. This suggests that these membrane insertion machineries all share a common ancestry. To test whether these ER proteins can functionally replace Oxa1 in yeast mitochondria, we generated strains that express mitochondria-targeted Get2–Get1 and Emc6–Emc3 fusion proteins in Oxa1 deletion mutants. Interestingly, the Emc6–Emc3 fusion was able to complement an Δoxa1 mutant and restored its respiratory competence. The Emc6–Emc3 fusion promoted the insertion of the mitochondrially encoded protein Cox2, as well as of nuclear encoded inner membrane proteins, although was not able to facilitate the assembly of the Atp9 ring. Our observations indicate that protein insertion into the ER is functionally conserved to the insertion mechanism in bacteria and mitochondria and adheres to similar topological principles.

Redirecting the core subunits of the protein membrane insertion complex EMC into mitochondria rescues cells deficient for the mitochondrial Oxa1 system; this supports the hypothesis that the machinery for protein insertion into the ER membrane is functionally analogous to the YidC/Oxa1/Alb3 family of bacteria, mitochondria and chloroplasts.     

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.     

Conserved mechanism of Oxa1 insertion into the mitochondrial inner membrane

Oxa1 is the mitochondrial representative of a family of related proteins that mediate the insertion of substrate proteins into the membranes of bacteria, chloroplasts, and mitochondria. Several studies have demonstrated that the bacterial homologue YidC participates both in the direct uptake of proteins from the bacterial cytosol, and in the uptake of nascent proteins from the Sec translocase. Studies on the biogenesis of membrane proteins in mitochondria established that Oxa1 has the capability to receive substrates at the inner surface of the inner membrane. In this study, we asked if Oxa1 may similarly cooperate with a protein translocase within the membrane. Since Oxa1 is involved in its own biogenesis, we used the precursor of Oxa1 as a model protein and investigated its import pathway. We found that immediately after import into mitochondria, Oxa1 initially accumulates at Tim23 that forms the inner membrane protein translocase. Cleavage of the Oxa1 presequence is dependent on mtHsp70, a heat shock protein of the mitochondrial matrix. However, mutant mtHsp70 showing a defect in the release of bound substrate proteins does not interfere with subsequent membrane insertion, indicating that membrane insertion of the mature protein is essentially mtHsp70-independent. We conclude that Oxa1 has the ability to accept preproteins within the membrane.     

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.     

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.     

Topogenesis of Mammalian Oxa1, a Component of the Mitochondrial Inner Membrane Protein Export Machinery

Oxa1 is a mitochondrial inner membrane protein with a predicted five-transmembrane segment (TM1∼5) topology in which the N terminus and a hydrophilic loop, L2, are exposed to the intermembrane space and the C-terminal region and two loops, L1 and L3, are exposed to the matrix. Oxa1 mediates the insertion of mitochondrial DNA-encoded subunits of respiratory complexes and several nuclear DNA-encoded proteins into the inner membrane from the matrix. Compared with yeast Oxa1, little is known about the import and function of mammalian Oxa1. Here, we investigated the topogenesis of Oxa1 in HeLa cells using systematic deletion or mutation constructs and found that (i) the N-terminal 64-residue segment formed a presequence, and its deletion directed the mature protein to the endoplasmic reticulum, indicating that the presequence arrests cotranslational activation of the potential endoplasmic reticulum-targeting signal within mature Oxa1, (ii) systematic deletion of Oxa1 TM segments revealed that the presence of all five TMs is essential for efficient membrane integration, (iii) the species-conserved hexapeptide (GLPWWG) located near the N terminus of TM1 was essential for export of the N-terminal segment and L2 into the intermembrane space from the matrix, i.e. for correct topogenesis of Oxa1, and (iv) GLPWWG placed near the N terminus of TM2 or TM3 in the reporter construct also supported its membrane integration in the Nout-Cin orientation. Together, these results demonstrated that topogenesis of Oxa1 is a cooperative event of all five TMs, and GLPWWG followed immediately by TM1 is essential for correct Oxa1 topogenesis.Most mitochondrial proteins are nuclear DNA-coded, and their import into mitochondrial compartments, that is, the mitochondrial outer membrane (MOM),³ mitochondrial inner membrane (MIM), intermembrane space (IMS), and matrix, is mediated by five protein translocation systems: translocase of the outer membrane (TOM complex), sorting and assembly machinery of MOM (SAM/TOB), translocases of the inner membrane (TIM23 complex and TIM22 complex), and a fifth system in the MIM that mediates integration of proteins from the matrix into the MIM (1, 2). The last system, which has been analyzed in detail in yeast, requires a membrane potential across the MIM and matrix ATP and mediates MIM integration of the mtDNA-encoded proteins as well as the integration of certain nuclear DNA-encoded proteins considered to be of bacterial origin, such as cytochrome c oxidase subunit II, F1Fo-ATPase subunit 9, and Oxa1 (3–5). Translocation efficiency is affected by the charge difference across the transmembrane (TM) in accordance with the positive-inside rule (5). Furthermore, the matrix-exposed C-terminal segment of Oxa1 is essential for binding mitochondrial ribosomes during cotranslational integration of mtDNA-encoded proteins (6, 7). Recent reports further demonstrated that the MIM protein Mba1, as a ribosome receptor, cooperates with the C-terminal ribosome binding segment of Oxa1 (8). The machinery and the underlying mechanisms of MIM insertion from the matrix must be further analyzed.Oxa1 protein, originally identified in yeast, is a component of the matrix-to-MIM export system conserved from prokaryote to eukaryote and is involved in Oxa1 biogenesis (9–14). YidC, a bacterial homologue of Oxa1, is involved in the biogenesis of inner membrane proteins in a Sec-dependent or Sec-independent manner (15, 16). In yeast, IMS export from the matrix of the Oxa1 N-terminal segment emerging from the Tim23 channel requires a membrane potential (4, 17), and the export is compromised in mitochondria isolated from a temperature-sensitive Oxa1-expressing strain at a non-permissive temperature (12). Herrmann and Bonnefoy (18) reported that Oxa1 protein functions in the export of a single hydrophilic loop region that was artificially produced by ligating the C-terminal region of cytochrome b with cytochrome c oxidase subunit II and placed between TM segments. Direct interaction of Oxa1 with an immature subunit in complex V was observed during its biogenesis (19). So far, these studies have only been performed in yeast, and no information is available on the mechanism of topogenesis in mammals with regard to how Oxa1 is involved in the export of multiple regions in a protein molecule. Our in vivo study revealed that the correct topogenesis of Oxa1 in the MIM proceeds as a result of the cooperation of all five TMs and that the cooperation of TM1 and the species-conserved six-residue segment (GLPWWG) in the N-terminal flanking region is essential for export from the matrix of both the N-terminal segment and hydrophilic L2 into the IMS.     

Chloroplast YidC homolog Albino3 can functionally complement the bacterial YidC depletion strain and promote membrane insertion of both bacterial and chloroplast thylakoid proteins

A new component of the bacterial translocation machinery, YidC, has been identified that specializes in the integration of membrane proteins. YidC is homologous to the mitochondrial Oxa1p and the chloroplast Alb3, which functions in a novel pathway for the insertion of membrane proteins from the mitochondrial matrix and chloroplast stroma, respectively. We find that Alb3 can functionally complement the Escherichia coli YidC depletion strain and promote the membrane insertion of the M13 procoat and leader peptidase that were previously shown to depend on the bacterial YidC for membrane translocation. In addition, the chloroplast Alb3 that is expressed in bacteria is essential for the insertion of chloroplast cpSecE protein into the bacterial inner membrane. Surprisingly, Alb3 is not required for the insertion of cpSecE into the thylakoid membrane. These results underscore the importance of Oxa1p homologs for membrane protein insertion in bacteria and demonstrate that the requirement for Oxa1p homologs is different in the bacterial and thylakoid membrane systems.     

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.     

YidC/Alb3/Oxa1 Family of Insertases

The YidC/Alb3/Oxa1 family functions in the insertion and folding of proteins in the bacterial cytoplasmic membrane, the chloroplast thylakoid membrane, and the mitochondrial inner membrane. All members share a conserved region composed of five transmembrane regions. These proteins mediate membrane insertion of an assorted group of proteins, ranging from respiratory subunits in the mitochondria and light-harvesting chlorophyll-binding proteins in chloroplasts to ATP synthase subunits in bacteria. This review discusses the YidC/Alb3/Oxa1 protein family as well as their function in membrane insertion and two new structures of the bacterial YidC, which suggest a mechanism for membrane insertion by this family of insertases.     

Oxa1-Ribosome Complexes Coordinate the Assembly of Cytochrome c Oxidase in Mitochondria

The terminal enzyme of the respiratory chain, cytochrome c oxidase, consists of a hydrophobic reaction center formed by three mitochondrially encoded subunits with which 9–10 nuclear encoded subunits are associated. The three core subunits are synthesized on mitochondrial ribosomes and inserted into the inner membrane in a co-translational reaction facilitated by the Oxa1 insertase. Oxa1 consists of an N-terminal insertase domain and a C-terminal ribosome-binding region. Mutants lacking the C-terminal region show specific defects in co-translational insertion, suggesting that the close contact of the ribosome with the insertase promotes co-translational insertion of nascent chains. In this study, we inserted flexible linkers of 100 or 200 amino acid residues between the insertase domain and ribosome-binding region of Oxa1 of Saccharomyces cerevisiae. In the absence of the ribosome receptor Mba1, these linkers caused a length-dependent decrease in mitochondrial respiratory activity caused by diminished levels of cytochrome c oxidase. Interestingly, considerable amounts of mitochondrial translation products were still integrated into the inner membrane in these linker mutants. However, they showed severe defects in later stages of the biogenesis process, presumably during assembly into functional complexes. Our observations suggest that the close proximity of Oxa1 to ribosomes is not only used to improve membrane insertion but is also critical for the productive assembly of the subunits of the cytochrome c oxidase. This points to a role for Oxa1 in the spatial coordination of the ribosome with assembly factors that are critical for enzyme biogenesis.     

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.     

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.     

Mba1, a membrane-associated ribosome receptor in mitochondria

The genome of mitochondria encodes a small number of very hydrophobic polypeptides that are inserted into the inner membrane in a cotranslational reaction. The molecular process by which mitochondrial ribosomes are recruited to the membrane is poorly understood. Here, we show that the inner membrane protein Mba1 binds to the large subunit of mitochondrial ribosomes. It thereby cooperates with the C-terminal ribosome-binding domain of Oxa1, which is a central component of the insertion machinery of the inner membrane. In the absence of both Mba1 and the C-terminus of Oxa1, mitochondrial translation products fail to be properly inserted into the inner membrane and serve as substrates of the matrix chaperone Hsp70. We propose that Mba1 functions as a ribosome receptor that cooperates with Oxa1 in the positioning of the ribosome exit site to the insertion machinery of the inner membrane.     

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.     

Analysis of 953 Human Proteins from a Mitochondrial HEK293 Fraction by Complexome Profiling

Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification – mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP.