Evolution of YidC/Oxa1/Alb3 insertases: three independent gene duplications followed by functional specialization in bacteria, mitochondria and chloroplasts

Members of the YidC/Oxa1/Alb3 protein family facilitate the insertion, folding and assembly of proteins of the inner membranes of bacteria and mitochondria and the thylakoid membrane of plastids. All homologs share a conserved hydrophobic core region comprising five transmembrane domains. On the basis of phylogenetic analyses, six subgroups of the family can be distinguished which presumably arose from three independent gene duplications followed by functional specialization. During evolution of bacteria, mitochondria and chloroplasts, subgroup-specific regions were added to the core domain to facilitate the association with ribosomes or other components contributing to the substrate spectrum of YidC/Oxa1/Alb3 proteins.     

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.     

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.     

Arabidopsis thaliana Oxa proteins locate to mitochondria and fulfill essential roles during embryo development

Members of the Alb3/Oxa1/YidC protein family function as insertases in chloroplasts, mitochondria, and bacteria. Due to independent gene duplications, all organisms possess two isoforms, Oxa1 and Oxa2 except gram-negative bacteria, which encode only for one YidC-like protein. The genome of Arabidopsis thaliana however, encodes for eight different isoforms. The localization of three of these isoforms has been identified earlier: Alb3 and Alb4 located in thylakoid membranes of chloroplasts while AtOxa1 was found in the inner membrane of mitochondria. Here, we show that the second Oxa1 protein, Oxa1b as well as two Oxa2 proteins are also localized in mitochondria. The last two isoforms most likely encode truncated versions of Oxa-like proteins, which might be inoperable pseudogenes. Homozygous mutant lines were only obtained for Oxa1b, which did not reveal any significant phenotypes, while T-DNA insertion lines of Oxa1a, Oxa2a and Oxa2b resulted only in heterozygous plants indicating that these genes are indispensable for plant development. Phenotyping heterozygous lines showed that embryos are either retarded in growth, display an albino phenotype or embryo formation was entirely abolished suggesting that Oxa1a and both Oxa2 proteins function in embryo formation although at different developmental stages as indicated by the various phenotypes observed.     

Analysis of the pdx-1 (snz-1/sno-1) region of the Neurospora crassa genome: correlation of pyridoxine-requiring phenotypes with mutations in two structural genes

We report the analysis of a 36-kbp region of the Neurospora crassa genome, which contains homologs of two closely linked stationary phase genes, SNZ1 and SNO1, from Saccharomyces cerevisiae. Homologs of SNZ1 encode extremely highly conserved proteins that have been implicated in pyridoxine (vitamin B6) metabolism in the filamentous fungi Cercospora nicotianae and in Aspergillus nidulans. In N. crassa, SNZ and SNO homologs map to the region occupied by pdx-1 (pyridoxine requiring), a gene that has been known for several decades, but which was not sequenced previously. In this study, pyridoxine-requiring mutants of N. crassa were found to possess mutations that disrupt conserved regions in either the SNZ or SNO homolog. Previously, nearly all of these mutants were classified as pdx-1. However, one mutant with a disrupted SNO homolog was at one time designated pdx-2. It now appears appropriate to reserve the pdx-1 designation for the N. crassa SNZ homolog and pdx-2 for the SNO homolog. We further report annotation of the entire 36,030-bp region, which contains at least 12 protein coding genes, supporting a previous conclusion of high gene densities (12,000-13,000 total genes) for N. crassa. Among genes in this region other than SNZ and SNO homologs, there was no evidence of shared function. Four of the genes in this region appear to have been lost from the S. cerevisiae lineage.     

The priB gene of Klebsiella pneumoniae encodes a 104-amino acid protein that is similar in structure and function to Escherichia coli PriB

Primosome protein PriB is a single-stranded DNA-binding protein that serves as an accessory factor for PriA helicase-catalyzed origin-independent reinitiation of DNA replication in bacteria. A recent report describes the identification of a novel PriB protein in Klebsiella pneumoniae that is significantly shorter than most sequenced PriB homologs. The K. pneumoniae PriB protein is proposed to comprise 55 amino acid residues, in contrast to E. coli PriB which comprises 104 amino acid residues and has a length that is typical of most sequenced PriB homologs. Here, we report results of a sequence analysis that suggests that the priB gene of K. pneumoniae encodes a 104-amino acid PriB protein, akin to its E. coli counterpart. Furthermore, we have cloned the K. pneumoniae priB gene and purified the 104-amino acid K. pneumoniae PriB protein. Gel filtration experiments reveal that the K. pneumoniae PriB protein is a dimer, and equilibrium DNA binding experiments demonstrate that K. pneumoniae PriB's single-stranded DNA-binding activity is similar to that of E. coli PriB. These results indicate that the PriB homolog of K. pneumoniae is similar in structure and in function to that of E. coli.     

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.     

Characterization of a novel bacterial arginine kinase from Desulfotalea psychrophila

Phosphagen kinases are found throughout the animal kingdom and catalyze the transfer of a high-energy gamma phosphoryl-group from ATP to a guanidino group on a suitable acceptor molecule such as creatine or arginine. Recent genome sequencing efforts in several proteobacteria, including Desulfotalea psychrophila LSv54, Myxococcus xanthus, Sulfurovum sp. NBC37-1, and Moritella sp. PE36 have revealed what appears to be a phosphagen kinase homolog present in their genomes. Based on sequence comparisons these putative homologs bear a strong resemblance to arginine kinases found in many invertebrates and some protozoa. We describe here a biochemical characterization of one of these homologs from D. psychrophila expressed in E. coli that confirms its ability to reversibly catalyze phosphoryl transfer from ATP to arginine. A phylogenetic analysis suggests that these bacteria homologs are not widely distributed in proteobacteria species. They appear more related to protozoan arginine kinases than to similar proteins seen in some Gram-positive bacteria that share key catalytic residues but encode protein tyrosine kinases. This raises the possibility of horizontal gene transfer as a likely origin of the bacterial arginine kinases.     

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 (approximately 90 amino acid residues) is translocated back across the inner membrane into the intermembrane space and thereby attains its native N(out)-C(in) 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.     

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.     

Identification of genes in the genome of the archaeon Methanosarcina mazeii that code for homologs of nuclear eukaryotic molecules involved in RNA processing

A 2.6kb fragment of chromosomal DNA from the archaeon Methanosarcina mazeii was sequenced and analyzed, and it was found to contain coding regions for three proteins that were 321, 234, and 193 amino acids (aa) in length. Homologs of the 321-aa protein were found in all archaeal genomes examined, but not in eukaryotic or bacterial genomes, with one exception in the latter. The protein with 234aa (named PrpM) was most similar to the putative protein Prp31p from Methanobacterium thermoautotrophicum, while the 193-aa protein (named FibM) was identified as an archaeal fibrillarin homolog. Prp and fibrillarin proteins are involved in RNA processing in eukaryotes, but their functions in archaea are not yet understood. The M. mazeii PrpM was also similar to three proteins from Saccharomyces cerevisiae: Prp31p, Nop56p, and Nop58p. Prp31p is a pre-mRNA processing protein, while Nop56p and Nop58p are involved in rRNA processing and interact with fibrillarin. No homologs of either protein were found in bacteria. The archaeal fibrillarin was shorter than its eukaryotic counterpart because it lacked the N-terminal glycine-arginine-rich (GAR) domain, present in most eukaryal homologs. The archaeal prp and fibrillarin gene homologs were found adjacent to each other, whereas in eukarya these genes are on separate chromosomes. Sequence signatures typical of the eukaryal molecules were identified in the M. mazeii and the other archaeal molecules studied. The close proximity of the prp and fib genes raises the possibility of a Prp-fibrillarin interaction in archaea.     

Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes

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 K_d 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).     

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.     

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.     

Characterization of the nuclear- and plastid-encoded secA-homologous genes in the unicellular red alga Cyanidioschyzon merolae

SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.     

A possible role of Atg8 homologs as a scaffold for signal transduction

Atg8 and its homologs are essential for autophagosome formation in various species. In animal cells, Atg8 homologs have an additional function in clearance of damaged organelles and bacteria, acting as a landmark for selective autophagy. We have recently shown that OATL1, a Rab-GTPase-activating protein (Rab-GAP), is a novel binding partner of Atg8 homologs in mammalian cells, but to our surprise, it is not a substrate of autophagy. Further analysis indicates that OATL1 is involved in the fusion between autophagosomes and lysosomes through its GAP activity and its Atg8 homolog binding activity. Our findings suggest a novel function of Atg8 homologs as a scaffold for signal transduction that regulates autophagosomal maturation.     

A possible role of Atg8 homologs as a scaffold for signal transduction

Atg8 and its homologs are essential for autophagosome formation in various species. In animal cells, Atg8 homologs have an additional function in clearance of damaged organelles and bacteria, acting as a landmark for selective autophagy. We have recently shown that OATL1, a Rab-GTPase-activating protein (Rab-GAP), is a novel binding partner of Atg8 homologs in mammalian cells, but to our surprise, it is not a substrate of autophagy. Further analysis indicates that OATL1 is involved in the fusion between autophagosomes and lysosomes through its GAP activity and its Atg8 homolog binding activity. Our findings suggest a novel function of Atg8 homologs as a scaffold for signal transduction that regulates autophagosomal maturation.     

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.     

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.     

The Alb3/Oxa1/YidC protein family: membrane-localized chaperones facilitating membrane protein insertion?

The recently identified Alb3/Oxa1/YidC family constitutes a novel class of proteins that function in promoting membrane insertion in chloroplasts, mitochondria and bacteria. These proteins mediate membrane insertion of a diverse group of membrane proteins that range from phage coat proteins in bacteria and respiratory-chain protein subunits in mitochondria to the light-harvesting chlorophyll-binding proteins in chloroplasts. Here, we discuss the Alb3/Oxa1/YidC protein family and their possible function as membrane chaperones, helping newly synthesized proteins to fold into the membrane bilayer.