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* This work was supported, in whole or in part, by National Institutes of Health Grants GM 32734 (to L. L. S.) and GM071034 (to E. C. K.). The on-line version of this article (available at http://www.jbc.org) contains supplemental Materials and Methods and Tables S1–S3 and Figs. S1–S4.
In humans the mitochondrial inner membrane protein Oxa1L is involved in the biogenesis of membrane proteins and facilitates the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane. The C-terminal ∼100-amino acid tail of Oxa1L (Oxa1L-CTT) binds to mitochondrial ribosomes and plays a role in the co-translational insertion of mitochondria-synthesized proteins into the inner membrane. Contrary to suggestions made for yeast Oxa1p, our results indicate that the C-terminal tail of human Oxa1L does not form a coiled-coil helical structure in solution. The Oxa1L-CTT exists primarily as a monomer in solution but forms dimers and tetramers at high salt concentrations. The binding of Oxa1L-CTT to mitochondrial ribosomes is an enthalpy-driven process with a Kd of 0.3–0.8 μm and a stoichiometry of 2. Oxa1L-CTT cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins L13, L20, and L28 and to mammalian mitochondrial specific ribosomal proteins MRPL48, MRPL49, and MRPL51. Oxa1L-CTT does not cross-link to proteins decorating the conventional exit tunnel of the bacterial large ribosomal subunit (L22, L23, L24, and L29).
Mammalian mitochondria synthesize 13 proteins, all of which are inserted into the respiratory chain complexes in the inner membrane. These hydrophobic polypeptides are integrated into the membrane during or immediately after their synthesis on mitochondrial ribosomes. Members of the Oxa1 evolutionarily conserved family of proteins including mammalian mitochondrial Oxa1L, yeast mitochondrial Oxa1p, chloroplast Alb3, and bacterial YidC are involved in the insertion of hydrophobic proteins into membranes (
). Both of these proteins bind to yeast mitochondrial ribosomes and facilitate the insertion of mitochondrial translation products into the inner membrane. Mammalian systems have a clear homolog of Oxa1p (Oxa1L). Mammals do not have a convincing member of the Mba1 family, although it has been suggested that the role of yeast Mba1 may be played by ribosomal protein MRPL45 in mammals (
A number of studies have been carried out on yeast and Neurospora crassa Oxa1p. These proteins contain an N-terminal region located in the intermembrane space, 5 transmembrane helices, and a C-terminal tail of about 90 amino acids located in the mitochondrial matrix (
) (Fig. 1A). Genetic and biochemical studies indicate that the C-terminal tail binds to mitochondrial ribosomes and plays an important role in the insertion of newly synthesized mitochondrial polypeptides into the inner membrane (
). Cross-linking studies indicate that yeast Oxa1p is located close to Mrp20 (a homolog of bacterial L23) and MrpL40 (the homolog of L24), both of which are located near the exit tunnel on the large subunit (
). Mammalian mitochondrial ribosomes are composed of only 25–30% RNA contributed by two rRNAs, 12 S in the small subunit and 16 S in the large subunit. The bulk of the mammalian mitochondrial ribosome consists of proteins (
). Mammalian mitochondrial ribosomes are 55 S particles and have ∼79 proteins, of which 42 have homologs in prokaryotic ribosomes, whereas 35 are specific for mitochondrial ribosomes. Twenty of these proteins have no clear homologs in yeast mitochondrial ribosomes (
), indicating a significant divergence between the protein composition of mitochondrial ribosomes from higher and lower eukaryotes. These differences may lead to significant alterations in the interaction of mitochondrial ribosomes from different sources with the machinery required for the insertion of mitochondrial translation products into the inner membrane.
) recently reported that human Oxa1L exists as a 600–700-kDa heterooligomeric complex in mitochondria from human embryonic kidney cells. Knockdown of human Oxa1L damages the biogenesis of the F1F0-ATP synthase and of Complex I (NADH:ubiquinone oxidoreductase) without altering the content of Complexes III or IV. These effects are distinct from those observed in yeast in which the assembly of Complex IV is strongly affected by mutations in Oxa1p (
). In the current report, we have examined the structure of the C-terminal tail of human Oxa1L (Oxa1L-CTT) and analyzed its interaction with the mitochondrial ribosome.
The Oxa1 family of proteins including yeast Oxa1p, chloroplast Abl3, and bacterial YidC is widely distributed in nature. Much remains to be learned about the mechanism of action of Oxa1 in the insertion of proteins into membranes. Most of the studies on the role of this protein in mitochondria have been carried out in yeast. In this organism the C-terminal tail has been shown to play an important role in binding to the large subunit of the ribosome and in promoting the insertion of mitochondrial translation products into the inner membrane. Experiments using CD suggested that the C-terminal tail of yeast Oxa1p forms a coiled-coil structure (
). Recent studies indicate that yeast Oxa1p and E. coli YidC exist as monomers, dimers, and higher oligomers at low detergent concentrations, whereas increasing detergent concentrations promote the formation of mostly monomers (
). The role of the C-terminal tail of Oxa1 in the oligomerization state is unclear. The C-terminal tail of human Oxa1L is largely monomeric in solution but does tend to oligomerize at increasing salt concentrations. However, like yeast Oxa1p, the data presented here indicate that two molecules of human Oxa1L-CTT bind to a single ribosome.
It is clear from studies of yeast Oxa1p that the C-terminal tail is necessary for ribosome binding and that this binding is independent of the presence of a nascent chain on the ribosome (
Our data indicate that human Oxa1L-CTT binds to mammalian 55 S ribosomes quite differently from that observed in the interaction of yeast Oxa1p with either yeast mitochondrial 74 S ribosomes or bacterial 70 S ribosomes. One striking difference between prokaryotic and mammalian mitochondrial ribosomes is found in the region of the large subunit corresponding to the exit tunnel. In bacterial ribosomes, this region contains large amounts of rRNA from domains I and III of the 23 S rRNA. Yeast Oxa1p contacts helices H24 and H59 when it is bound to E. coli ribosomes (
). Yeast mitochondrial ribosomes have a segment equivalent to H24 and a region encompassing H59, although secondary structure predictions of this region of the large subunit rRNA do not correspond to the structure observed in E. coli 23 S rRNA. In contrast, the large subunit rRNA of mammalian mitochondria (16 S rRNA) has almost completely lost the regions of the rRNA corresponding to domains I and III. Both helices H24 and H59 are missing in mammalian mitochondrial ribosomes, suggesting that mammalian Oxa1L will interact with ribosomes in quite a different way.
In addition to large amounts of rRNA, several proteins are located at or near the exit tunnel of bacterial ribosomes including L22, L23, L24, and L29 (Fig. 7A). Homologs of all of these proteins are present in yeast mitochondrial ribosomes. In addition, the region near the exit tunnel of yeast mitochondrial ribosomes contains several mitochondrial specific ribosomal proteins including Mrpl3, Mrpl13, and Mrpl27 (
). One site corresponds to the traditional exit site located about 88 Å from the peptidyltransferase site. The other, termed the polypeptide accessible site, is located closer (about 68 Å from the peptidyltransferase). It is possible that some nascent chains may emerge from the large subunit through the polypeptide accessible site. Both of these regions are dominated by ribosomal proteins rather than rRNA (
). Ribosomal proteins corresponding to bacterial L22, L23, and L24 are present in mammalian mitochondrial ribosomes, but significant fractions of these proteins are obscured by mitochondrial-specific ribosomal proteins. There is no clear homolog of L29, although it has been suggested that MRPL47 may be a distant ortholog of L29 (
). Two of the mitochondrial specific ribosomal proteins found near the exit tunnel in yeast are observed in mammalian mitochondrial ribosomes; yeast Mrpl27 is homologous to mammalian mitochondrial MRPL41, and yeast Mrpl3 corresponds to MRPL44. No homolog of yeast Mrpl13, found near the exit tunnel, has been observed in mammalian mitochondrial ribosomes. Despite some similarity in the protein composition between yeast and mammalian mitochondrial ribosomes, very different ribosomal partner proteins were observed for human Oxa1L-CTT than seen with yeast Oxa1p. Neither MRPL23 nor MRPL24 was observed to cross-link to human Oxa1L-CTT. In some respects this observation is surprising as they cross-link to yeast Oxa1p. However, large portions of MRPL23 and MRPL24 are covered by mitochondrial-specific ribosomal proteins in mammals (
) potentially making them inaccessible for cross-linking to Oxa1L.
Oxa1L-CTT is cross-linked to homologs of bacterial ribosomal protein L13, L20, and L28 (Fig. 7B). These homologs are all located on the solvent side of the large subunit spanning both sides of the exit tunnel and lie somewhat above the tunnel (Fig. 7B). MRPL28 is considerably larger than its bacterial homolog (Fig. 7C) and may cover a significant fraction of the back of the subunit. Furthermore, the lack of significant portions of Domains I and III of the large subunit rRNA in mitochondrial 39 S subunits exposes a larger portion of this protein on the solvent side of the ribosome (Fig. 7B). It is noteworthy that MRPL20, which cross-links to human Oxa1L-CTT, is missing in yeast mitochondrial ribosomes.
In addition to the bacterial homologs, three mitochondrial-specific proteins (MRPL48, MRPL49, and MRPL51) were identified close to Oxa1L-CTT. Neither MRPL48 nor MRPL51 has a homolog in yeast. The locations of MRPL48, MRPL49, and MRPL51 are not known, but the yeast homolog of MRPL49 (Img2) has been reported to interact with the homologs of L23 and L19 placing it on the solvent side of the large subunit and relatively close to the exit tunnel in mitochondrial ribosomes (
). Of six proteins cross-linked to human Oxa1L-CTT, three are missing in yeast (MRPL20, MRPL48, and MRPL51). Therefore, the decoration of the exit tunnel and the interaction of Oxa1 is clearly different between yeast and mammalian mitochondrial ribosomes.
Although we cannot rule out the possibility that other ribosomal proteins will cross-link to the full-length Oxa1L when it is organized in the membrane, the data presented here indicate that Oxa1L-CTT binds to the solvent side of the large subunit where it is located close to the exit tunnel and to a number of proteins that have no bacterial homologs. These proteins may play a role in the interaction of the mitochondrial ribosome with the nascent chain and may facilitate the integration of mitochondrial translation products into the respiratory chain complexes into the inner membrane.
We thank Dr. Ramesh Jha for creating the three-dimensional structure of Oxa1L-CTT based on de novo structure prediction protocols. We thank Dr. Carol Parker for technical assistance with the mass spectrometry results.