Oxal/Alb3/YidC system for insertion of membrane proteins in mitochondria,chloroplasts and bacteria (Review)

Recent studies have shown that there is a pathway that is evolutionarily conserved for the insertion of proteins into the membrane in mitochondria, chloroplasts, and bacteria. In this pathway, the Oxa1/Alb3/YidC proteins are believed to function as membrane insertases that play an important role in the membrane protein biogenesis of respiratory and energy transduction proteins. Additional roles of the Oxa1/Alb3/YidC members may be in the lateral integration of proteins into the lipid bilayer, and in the folding and assembly of proteins into membrane protein complexes.     

YidC/Oxa/Alb3蛋白家族

YidC/Oxa/Alb3蛋白家族是进化上保守的蛋白质转运酶,分别负责将一些与能量合成有关的膜蛋白转运到细菌内膜、线粒体内膜和叶绿体类囊体膜。它们具有保守的跨膜结构域,广泛存在于三界生物中。本文主要综述近年来对YidC/Oxa/Alb3家族成员的分布、结构、功能及进化的研究进展。     

YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins

Members of the YidC family exist in all three domains of life, where they control the assembly of a large variety of membrane protein complexes that function as transporters, energy devices, or sensor proteins. Recent studies in bacteria have shown that YidC functions on its own as a membrane protein insertase independent of the Sec protein-conducting channel. YidC can also assist in the lateral integration and folding of membrane proteins that insert into the membrane via the Sec pathway.     

A ribosome–nascent chain sensor of membrane protein biogenesis in Bacillus subtilis

Proteins in the YidC/Oxa1/Alb3 family have essential functions in membrane protein insertion and folding. Bacillus subtilis encodes two YidC homologs, one that is constitutively expressed (spoIIIJ/yidC1) and a second (yqjG/yidC2) that is induced in spoIIIJ mutants. Regulated induction of yidC2 allows B. subtilis to maintain capacity of the membrane protein insertion pathway. We here show that a gene located upstream of yidC2 (mifM/yqzJ) serves as a sensor of SpoIIIJ activity that regulates yidC2 translation. Decreased SpoIIIJ levels or deletion of the MifM transmembrane domain arrests mifM translation and unfolds an mRNA hairpin that otherwise blocks initiation of yidC2 translation. This regulated translational arrest and yidC2 induction require a specific interaction between the MifM C‐terminus and the ribosomal polypeptide exit tunnel. MifM therefore acts as a ribosome–nascent chain complex rather than as a fully synthesized protein. B. subtilis MifM and the previously described secretion monitor SecM in Escherichia coli thereby provide examples of the parallel evolution of two regulatory nascent chains that monitor different protein export pathways by a shared molecular mechanism.     

M13 procoat protein insertion into YidC and SecYEG proteoliposomes and liposomes

M13 procoat protein was one of the first model proteins used to study bacterial membrane protein insertion. It contains a signal peptide of 23 amino acid residues and is not membrane targeted by the signal recognition particle. The translocation of its periplasmic domain is independent of the preprotein translocase (SecAYEG) but requires electrochemical membrane potential and the membrane insertase YidC of Escherichia coli. We show here that YidC is sufficient for efficient membrane insertion of the purified M13 procoat protein into energized YidC proteoliposomes. When no membrane potential is applied, the insertion is substantially reduced. Only in the presence of YidC, membrane insertion occurs if bilayer integrity is preserved and membrane potential is stable for more than 20 min. A mutant of the M13 procoat protein, H5EE, with two additional negatively charged residues in the periplasmic domain inserted into YidC proteoliposomes and SecYEG proteoliposomes with equal efficiencies. We conclude that the protein can use both the YidC-only pathway and the Sec pathway. This poses the questions of how procoat H5EE is inserted in vivo and how insertion pathways are selected in the cell.     

Membrane biogenesis of subunit II of cytochrome bo oxidase: contrasting requirements for insertion of N-terminal and C-terminal domains

The membrane assembly of the respiratory complexes requires the membrane insertases Oxa1 in mitochondria and YidC in bacteria. Oxa1 is responsible for the insertion of the mitochondrial cytochrome c oxidase subunit II (CoxII). Here, we investigated whether YidC, the bacterial Oxa1 homolog, plays a crucial role in the assembly of the bacterial subunit II (CyoA) of cytochrome bo oxidase. CyoA spans the membrane twice and is made with a cleavable signal peptide. We find that translocation of the short N-terminal domain of CyoA is YidC-dependent. In contrast, both the SecA/SecYEG complex and YidC are required for translocation of the large C-terminal domain. By studying the N-terminal domain of CyoA alone, we find that translocation is unaffected when SecE is depleted, suggesting that the YidC insertase on its own catalyzes membrane insertion of the N-terminal region of CyoA. Strikingly, we find that the translocation of the N-terminal domain is a prerequisite for translocation of the C-terminal domain in the full-length CyoA protein because translocation of the large C-terminal domain alone in a truncated CyoA derivative was observed in the absence of YidC. This work shows that the distinct domains of CyoA have different translocation requirements (YidC only and Sec/YidC) and confirms that the membrane biogenesis of subunit II of cytochrome oxidase in bacteria and mitochondria have conserved features.     

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 Role of the Strictly Conserved Positively Charged Residue Differs among the Gram-positive,Gram-negative,and Chloroplast YidC Homologs

Recently, the structure of YidC2 from Bacillus halodurans revealed that the conserved positively charged residue within transmembrane segment one (at position 72) is located in a hydrophilic groove that is embedded in the inner leaflet of the lipid bilayer. The arginine residue was essential for the Bacillus subtilis SpoIIIJ (YidC1) to insert MifM and to complement a SpoIIIJ mutant strain. Here, we investigated the importance of the conserved positively charged residue for the function of the Escherichia coli YidC, Streptococcus mutans YidC2, and the chloroplast Arabidopsis thaliana Alb3. Like the Gram-positive B. subtilis SpoIIIJ, the conserved arginine was required for functioning of the Gram-positive S. mutans YidC2 and was necessary to complement the E. coli YidC depletion strain and to promote insertion of a YidC-dependent membrane protein synthesized with one but not two hydrophobic segments. In contrast, the conserved positively charged residue was not required for the E. coli YidC or the A. thaliana Alb3 to functionally complement the E. coli YidC depletion strain or to promote insertion of YidC-dependent membrane proteins. Our results also show that the C-terminal half of the helical hairpin structure in cytoplasmic loop C1 is important for the activity of YidC because various deletions in the region either eliminate or impair YidC function. The results here underscore the importance of the cytoplasmic hairpin region for YidC and show that the arginine is critical for the tested Gram-positive YidC homolog but is not essential for the tested Gram-negative and chloroplast YidC homologs.     

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex,the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.     

The C Terminus of the Alb3 Membrane Insertase Recruits cpSRP43 to the Thylakoid Membrane

The YidC/Oxa1/Alb3 family of membrane proteins controls the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we describe the molecular mechanisms underlying the interaction of Alb3 with the chloroplast signal recognition particle (cpSRP). The Alb3 C-terminal domain (A3CT) is intrinsically disordered and recruits cpSRP to the thylakoid membrane by a coupled binding and folding mechanism. Two conserved, positively charged motifs reminiscent of chromodomain interaction motifs in histone tails are identified in A3CT that are essential for the Alb3-cpSRP43 interaction. They are absent in the C-terminal domain of Alb4, which therefore does not interact with cpSRP43. Chromodomain 2 in cpSRP43 appears as a central binding platform that can interact simultaneously with A3CT and cpSRP54. The observed negative cooperativity of the two binding events provides the first insights into cargo release at the thylakoid membrane. Taken together, our data show how Alb3 participates in cpSRP-dependent membrane targeting, and our data provide a molecular explanation why Alb4 cannot compensate for the loss of Alb3. Oxa1 and YidC utilize their positively charged, C-terminal domains for ribosome interaction in co-translational targeting. Alb3 is adapted for the chloroplast-specific Alb3-cpSRP43 interaction in post-translational targeting by extending the spectrum of chromodomain interactions.     

Interaction studies between the chloroplast signal recognition particle subunit cpSRP43 and the full-length translocase Alb3 reveal a membrane-embedded binding region in Alb3 protein

Posttranslational targeting of the light-harvesting chlorophyll a,b-binding proteins depends on the function of the chloroplast signal recognition particle, its receptor cpFtsY, and the translocase Alb3. The thylakoid membrane protein Alb3 of Arabidopsis chloroplasts belongs to the evolutionarily conserved YidC/Oxa1/Alb3 protein family; the members of this family facilitate the insertion, folding, and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here, we analyzed the interaction sites of full-length Alb3 with the cpSRP pathway component cpSRP43 by using in vitro and in vivo studies. Bimolecular fluorescence complementation and Alb3 proteoliposome studies showed that the interaction of cpSRP43 is dependent on a binding domain in the C terminus of Alb3 as well as an additional membrane-embedded binding site in the fifth transmembrane domain (TMD5) of Alb3. The C-terminal binding domain was mapped to residues 374-388, and the binding domain within TMD5 was mapped to residues 314-318 located close to the luminal end of TMD5. A direct binding between cpSRP43 and these binding motifs was shown by pepspot analysis. Further studies using blue-native gel electrophoresis revealed that full-length Alb3 is able to form dimers. This finding and the identification of a membrane-embedded cpSRP43 binding site in Alb3 support a model in which cpSRP43 inserts into a dimeric Alb3 translocation pore during cpSRP-dependent delivery of light-harvesting chlorophyll a,b-binding proteins.     

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.     

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.     

Oxa1/Alb3/YidC system for insertion of membrane proteins in mitochondria, chloroplasts and bacteria (review)

Recent studies have shown that there is a pathway that is evolutionarily conserved for the insertion of proteins into the membrane in mitochondria, chloroplasts, and bacteria. In this pathway, the Oxa1/Alb3/YidC proteins are believed to function as membrane insertases that play an important role in the membrane protein biogenesis of respiratory and energy transduction proteins. Additional roles of the Oxa1/Alb3/YidC members may be in the lateral integration of proteins into the lipid bilayer, and in the folding and assembly of proteins into membrane protein complexes.     

The Pf3 coat protein contacts TM1 and TM3 of YidC during membrane biogenesis

The coat protein of bacteriophage Pf3 is inserted into the plasma membrane of Escherichia coli by the insertase YidC. To identify which of the six transmembrane regions of YidC bind the single-spanning Pf3 coat protein during membrane protein biogenesis, we used the disulfide cross-linking approach. We generated single cysteines in each of the transmembrane regions of YidC and in the center of the hydrophobic region of Pf3 coat protein. We found that the substrate Pf3 coat contacts the first and third transmembrane segment (TM) of YidC as crosslinks between these two proteins can be formed in vivo during membrane biogenesis. A detailed disulfide-mapping study revealed that one face of TM3 of YidC makes contact with the Pf3 protein.

Structured summary

MINT-6795850, MINT-6795869, MINT-6795912, MINT-6795927, MINT-6795942:

Coat protein (uniprotkb:P03623) binds (MI:0408) YidC (uniprotkb:P25714) by cross-linking studies (MI:0030)

MINT-6795898:

Coat protein (uniprotkb:P03623) binds (MI:0408) Coat protein (uniprotkb:P03623) by cross-linking studies (MI:0030)

     

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 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.