CdsA, a CDP-diacylglycerol synthase involved in phospholipid and glycolipid MPIase biosynthesis, possesses multiple initiation codons.

CdsA is a CDP-diacylglycerol synthase essential for phospholipid and glycolipid MPIase biosynthesis, and therefore for growth. The initiation codon of CdsA has been assigned as "TTG," while methionine at the 37th codon was reported to be an initiation codon in the original report. Since a vector containing the open reading frame starting with "TTG" under a controllable promoter complemented the cdsA knockout, "TTG" could function as an initiation codon. However, no evidence supporting that this "TTG" is the sole initiation codon has been reported. We determined the initiation codon by examining the ability of mutants around the N-terminal region to complement cdsA mutants. Even if the "TTG" was substituted with a stop codon, the clear complementation was observed. Moreover, the clones with multiple mutations of stop codons complemented the cdsA mutant up to the 37th codon, indicating that cdsA possesses multiple codons that can function as initiation codons. We constructed an experimental system in which the chromosomal expression of cdsA can be analyzed. By means of this system, we found that the cdsA mutant with substitution of "TTG" with a stop codon is fully functional. Thus, we concluded that CdsA contains multiple initiation codons.

Coordinated upregulation of two CDP-diacylglycerol synthases, YnbB and CdsA, is essential for cell growth and membrane protein export in the cold.

YnbB is a paralogue of CdsA, a CDP-diacylglycerol synthase. While the cdsA gene is essential, the ynbB gene is dispensable. So far, no phenotype of ynbB knockout has been observed. We found that a ynbB knockout strain acquired cold-sensitivity on growth under CdsA-limited conditions. We found that MPIase, a glycolipid involved in protein export, is cold-upregulated to facilitate protein export in the cold, by increasing the mRNA levels of not only CdsA but also that of YnbB. Under non-permissive conditions, phospholipid biosynthesis proceeded normally, however, MPIase upregulation was inhibited with accumulation of precursors of membrane and secretory proteins such as M13 procoat and proOmpA, indicating that YnbB is dedicated to MPIase biosynthesis, complementing the CdsA function.

Structural Requirements of a Glycolipid MPIase for Membrane Protein Integration.

MPIase is a glycolipid involved in membrane protein integration in the inner membrane of Escherichia coli. To overcome the trace amounts and heterogeneity of natural MPIase, we systematically synthesized MPIase analogs. Structure-activity relationship studies revealed the contribution of distinctive functional groups and the effect of the MPIase glycan length on membrane protein integration activity. In addition, both the synergistic effects of these analogs with the membrane chaperone/insertase YidC, and the chaperone-like activity of the phosphorylated glycan were observed. These results verified the translocon-independent membrane integration mechanism in the inner membrane of E. coli, in which MPIase captures the highly hydrophobic nascent proteins via its characteristic functional groups, prevents protein aggregation, attracts the proteins to the membrane surface, and delivers them to YidC in order to regenerate its own integration activity.

A bacterial glycolipid essential for membrane protein integration.

The proper conformation and orientation of membrane protein integration in cells is an important biological event. Interestingly, a new factor named MPIase (membrane protein integrase) was proven essential in this process in Escherichia coli, besides proteinaceous factors, such as Sec translocons and an insertase YidC. A combination of spectroscopic analyses and synthetic work has revealed that MPIase is a glycolipid despite its enzyme-like activity. MPIase has a long glycan chain comprised of repeating trisaccharide units, a pyrophosphate linker, and a diacylglycerol anchor. In order to determine the mechanism of its activity, we synthesized a trisaccharyl pyrophospholipid termed mini-MPIase-3, a minimal unit of MPIase, and its derivatives. A significant activity of mini-MPIase-3 indicated that it involves an essential structure for membrane protein integration. We also analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate protein using physicochemical methods. The structure-activity relationship studies demonstrated that the glycan part of MPIase prevents the aggregation of substrate proteins, and the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles for interactions with substrate proteins. MPIase serves at an initial step in the Sec-independent integration, whereas YidC, proton motive force, and/or SecYEG cooperatively function(s) with MPIase at the following step in vivo. Furthermore, depletion of the biosynthetic enzyme demonstrated that MPIase is crucial for membrane protein integration and cell growth. Thus, we elucidated new biological functions of glycolipids using a combination of synthetic chemistry, biochemistry, physicochemical measurements, and molecular-biological approaches.

Interaction between glycolipid MPIase and proteinaceous factors during protein integration into the cytoplasmic membrane of E. coli.

Protein integration into biomembranes is an essential biological phenomenon common to all organisms. While various factors involved in protein integration, such as SRP, SecYEG and YidC, are proteinaceous, we identified a glycolipid named MPIase (Membrane Protein Integrase), which is present in the cytoplasmic membrane of E. coli. In vitro experiments using inverted membrane vesicles prepared from MPIase-depleted strains, and liposomes containing MPIase showed that MPIase is required for insertion of a subset of membrane proteins, which has been thought to be SecYEG-independent and YidC-dependent. Also, SecYEG-dependent substrate membrane proteins require MPIase in addition. Furthermore, MPIase is also essential for insertion of proteins with multiple negative charges, which requires both YidC and the proton motive force (PMF). MPIase directly interacts with SecYEG and YidC on the membrane. MPIase not only cooperates with these factors but also has a molecular chaperone-like function specific to the substrate membrane proteins through direct interaction with the glycan chain. Thus, MPIase catalyzes membrane insertion by accepting nascent membrane proteins on the membrane through its chaperone-like function, i.e., direct interaction with the substrate proteins, and then MPIase functionally interacts with SecYEG and YidC for substrate delivery, and acts with PMF to facilitate and complete membrane insertion when necessary. In this review, we will outline the mechanisms underlying membrane insertion catalyzed by MPIase, which cooperates with proteinaceous factors and PMF.

Role of a bacterial glycolipid in Sec-independent membrane protein insertion.

Non-proteinaceous components in membranes regulate membrane protein insertion cooperatively with proteinaceous translocons. An endogenous glycolipid in the Escherichia coli membrane called membrane protein integrase (MPIase) is one such component. Here, we focused on the Sec translocon-independent pathway and examined the mechanisms of MPIase-facilitated protein insertion using physicochemical techniques. We determined the membrane insertion efficiency of a small hydrophobic protein using solid-state nuclear magnetic resonance, which showed good agreement with that determined by the insertion assay using an in vitro translation system. The observed insertion efficiency was strongly correlated with membrane physicochemical properties measured using fluorescence techniques. Diacylglycerol, a trace component of E. coli membrane, reduced the acyl chain mobility in the core region and inhibited the insertion, whereas MPIase restored them. We observed the electrostatic intermolecular interactions between MPIase and the side chain of basic amino acids in the protein, suggesting that the negatively charged pyrophosphate of MPIase attracts the positively charged residues of a protein near the membrane surface, which triggers the insertion. Thus, this study demonstrated the ingenious approach of MPIase to support membrane insertion of proteins by using its unique molecular structure in various ways.

Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration.

Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.

Interplay between MPIase, YidC, and PMF during Sec-independent insertion of membrane proteins.

Integral membrane proteins with the N-out topology are inserted into membranes usually in YidC- and PMF-dependent manners. The molecular basis of the various dependencies on insertion factors is not fully understood. A model protein, Pf3-Lep, is inserted independently of both YidC and PMF, whereas the V15D mutant requires both YidC and PMF in vivo. We analyzed the mechanisms that determine the insertion factor dependency in vitro. Glycolipid MPIase was required for insertion of both proteins because MPIase depletion caused a significant defect in insertion. On the other hand, YidC depletion and PMF dissipation had no effects on Pf3-Lep insertion, whereas V15D insertion was reduced. We reconstituted (proteo)liposomes containing MPIase, YidC, and/or F0F1-ATPase. MPIase was essential for insertion of both proteins. YidC and PMF stimulated Pf3-Lep insertion as the synthesis level increased. V15D insertion was stimulated by both YidC and PMF irrespective of the synthesis level. These results indicate that charges in the N-terminal region and the synthesis level are the determinants of YidC and PMF dependencies with the interplay between MPIase, YidC, and PMF.

Expression of Cds4/5 of Arabidopsis chloroplasts in E. coli reveals the membrane topology of the C-terminal region of CDP-diacylglycerol synthases.

CDP-diacylglycerol synthases (Cds) are conserved from bacteria to eukaryotes. Bacterial CdsA is involved not only in phospholipid biosynthesis but also in biosynthesis of glycolipid MPIase, an essential glycolipid that catalyzes membrane protein integration. We found that both Cds4 and Cds5 of Arabidopsis chloroplasts complement cdsA knockout by supporting both phospholipid and MPIase biosyntheses. Comparison of the sequences of CdsA and Cds4/5 suggests a difference in membrane topology at the C-termini, since the region assigned as the last transmembrane region of CdsA, which follows the conserved cytoplasmic domain, is missing in Cds4/5. Deletion of the C-terminal region abolished the function, indicating the importance of the region. Both 6 × His tag attachment to CdsA and substitution of the C-terminal 6 residues with 6 × His did not affect the function. These 6 × His tags were sensitive to protease added from the cytosolic side in vitro, indicating that this region is not a transmembrane one but forms a membrane-embedded reentrant loop. Thus, the C-terminal region of Cds homologues forms a reentrant loop, of which structure is important for the Cds function.

Ring assembly of c subunits of F0 F1 -ATP synthase in Propionigenium modestum requires YidC and UncI following MPIase-dependent membrane insertion.

The c subunits of F0 F1 -ATP synthase (F0 c) assemble into a ring structure, following membrane insertion that is dependent on both glycolipid MPIase and protein YidC. We analyzed the insertion and assembly processes of Propionigenium modestum F0 c (Pm-F0 c), of which the ring structure is resistant to SDS. Ring assembly of Pm-F0 c requires P. modestum UncI (Pm-UncI). Ring assembly of in vitro synthesized Pm-F0 c was observed when both YidC and Pm-UncI were reconstituted into liposomes of Escherichia coli phospholipids. Under the physiological conditions where spontaneous insertion had been blocked by diacylglycerol, MPIase was necessary for Pm-F0 c insertion allowing the subsequent YidC/Pm-UncI-dependent ring assembly. Thus, we have succeeded in the complete reconstitution of membrane insertion and subsequent ring assembly of Pm-F0 c.

In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes.

It is important to experimentally determine how membrane proteins are integrated into biomembranes to unveil the roles of the integration factors, and to understand the functions and structures of membrane proteins. We have developed a reconstitution system for membrane protein integration in E. coli using purified factors, in which the integration reaction in vivo is highly reproducible. This system enabled not only analysis of membrane-embedded factors including glycolipid MPIase, but also elucidation of the detailed mechanisms underlying membrane protein integration. Using the system, the integration of membrane proteins can be evaluated in vitro through a protease-protection assay. We report here how to prepare (proteo)liposomes and to determine the activities of membrane protein integration.

Biosynthesis of glycolipid MPIase (membrane protein integrase) is independent of the genes for ECA (enterobacterial common antigen).

MPIase (membrane protein integrase) is an essential glycolipid that drives protein integration into the inner membrane of E. coli, while glycolipid ECA (enterobacterial common antigen) is a major component at the surface of the outer membrane. Irrespective of the differences in molecular weight, subcellular localization and function in cells, the glycan chains of the two glycolipids are similar, since the repeating unit comprising the glycan chains is the same. A series of biosynthetic genes for ECA, including ones for the corresponding nucleotide sugars, have been identified and extensively characterized. In this study, we found that knockouts as to the respective genes for ECA biosynthesis can grow in the minimum medium with the normal expression level of MPIase, indicating that MPIase can be biosynthesized de novo without the utilization of any compounds generated through ECA biosynthesis. Conversely, ECA was expressed normally upon MPIase depletion. From these results, we conclude that the biosynthetic genes for MPIase and ECA are independent.

The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins.

Bacterial membrane proteins are integrated into membranes through the concerted activities of a series of integration factors, including membrane protein integrase (MPIase). However, how MPIase activity is complemented by other integration factors during membrane protein integration is incompletely understood. Here, using inverted inner-membrane vesicle and reconstituted (proteo)liposome preparations from Escherichia coli cells, along with membrane protein integration assays and the PURE system to produce membrane proteins, we found that anti-MPIase IgG inhibits the integration of both the Sec-independent substrate 3L-Pf3 coat and the Sec-dependent substrate MtlA into E. coli membrane vesicles. MPIase-depleted membrane vesicles lacked both 3L-Pf3 coat and MtlA integration, indicating that MPIase is involved in the integration of both proteins. We developed a reconstitution system in which disordered spontaneous integration was precluded, which revealed that SecYEG, YidC, or both, are not sufficient for Sec-dependent and -independent integration. Although YidC had no effect on MPIase-dependent integration of Sec-independent substrates in the conventional assay system, YidC significantly accelerated the integration when the substrate amounts were increased in our PURE system-based assay. Similar acceleration by YidC was observed for MtlA integration. YidC mutants with amino acid substitutions in the hydrophilic cavity inside the membrane were defective in the acceleration of the Sec-independent integration. Of note, MPIase was up-regulated upon YidC depletion. These results indicate that YidC accelerates the MPIase-dependent integration of membrane proteins, suggesting that MPIase and YidC function sequentially and cooperatively during the catalytic cycle of membrane protein integration.

Alteration of Membrane Physicochemical Properties by Two Factors for Membrane Protein Integration.

After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.

Two-step induction of cdsA promoters leads to upregulation of the glycolipid MPIase at cold temperature.

Glycolipid MPIase, essential for membrane protein integration into the cytoplasmic membrane of Escherichia coli, is upregulated at cold temperatures. This upregulation is rapid and sustainable. CdsA, a CDP-diacylglycerol synthase, is a rate-limiting enzyme for MPIase biosynthesis. Upregulation of CdsA is responsible for the increase in the MPIase level at low temperature. Investigation of cdsA regulatory regions revealed at least two cold-inducible promoters, a cold-shock promoter that functions transiently and immediately in the cold, and one that is sustainable in the cold. The stability of the cdsA transcript was comparable with that of tufA, which is not cold-inducible. Thus, cdsA is induced through two-step cold-induction to maintain MPIase at a high level rapidly and sustainably in the cold.

Increased expression of the bacterial glycolipid MPIase is required for efficient protein translocation across membranes in cold conditions.

Protein integration into and translocation across biological membranes are vital events for organismal survival and are fundamentally conserved among many organisms. Membrane protein integrase (MPIase) is a glycolipid that drives membrane protein integration into the cytoplasmic membrane in Escherichia coli MPIase also stimulates protein translocation across the membrane, but how its expression is regulated is incompletely understood. In this study, we found that the expression level of MPIase significantly increases in the cold (<25 °C), whereas that of the SecYEG translocon does not. Using previously created gene-knockout E. coli strains, we also found that either the cdsA or ynbB gene, both encoding rate-limiting enzymes for MPIase biosynthesis, is responsible for the increase in the MPIase expression. Furthermore, using pulse-chase experiments and protein integration assays, we demonstrated that the increase in MPIase levels is important for efficient protein translocation, but not for protein integration. We conclude that MPIase expression is required to stimulate protein translocation in cold conditions and is controlled by cdsA and ynbB gene expression.

CdsA is involved in biosynthesis of glycolipid MPIase essential for membrane protein integration in vivo.

MPIase is a glycolipid that is involved in membrane protein integration. Despite evaluation of its functions in vitro, the lack of information on MPIase biosynthesis hampered verification of its involvement in vivo. In this study, we found that depletion of CdsA, a CDP-diacylglycerol synthase, caused not only a defect in phospholipid biosynthesis but also MPIase depletion with accumulation of the precursors of both membrane protein M13 coat protein and secretory protein OmpA. Yeast Tam41p, a mitochondrial CDP-diacylglycerol synthase, suppressed the defect in phospholipid biosynthesis, but restored neither MPIase biosynthesis, precursor processing, nor cell growth, indicating that MPIase is essential for membrane protein integration and therefore for cell growth. Consistently, we observed a severe defect in protein integration into MPIase-depleted membrane vesicles in vitro. Thus, the function of MPIase as a factor involved in protein integration was proven in vivo as well as in vitro. Moreover, Cds1p, a eukaryotic CdsA homologue, showed a potential for MPIase biosynthesis. From these results, we speculate the presence of a eukaryotic MPIase homologue.

Expression of alcohol oxidase gene from Ochrobactrum sp. AIU 033 in recombinant Escherichia coli through the twin-arginine translocation pathway.

We cloned a set of genes encoding alcohol oxidase from Ochrobactrum sp. AIU 033 (OcAOD), which exhibits the appropriate substrate specificity for glyoxylic acid production from glycolic acid. The set of genes for OcAOD contained two open reading frames consisting of 555-bp (aodB) and 1572-bp (aodA) nucleotides, which encode the precursor for the ß-subunit and α-subunit of OcAOD, respectively. We expressed the cloned genes as an active product in Escherichia coli BL21(DE3). The recombinant OcAOD oxidized glycolic acid and primary alcohols with C2-C8 but not glyoxylic acid (as is the case for native OcAOD), whereas the Km and Vmax values for glycolic acid and the pH stability were higher than those of native OcAOD. A consensus sequence for the twin-arginine translocation (Tat) pathway was identified in the N-terminal region of the precursor for the ß-subunit, and the active form of OcAOD was localized in the periplasm of recombinant E. coli, which indicated that OcAOD would be transported from the cytoplasm to the periplasm by the hitchhiker mechanism through the Tat pathway. The OcAOD productivity of the recombinant E. coli was 24-fold higher than that of Ochrobactrum sp. AIU 033, and it was further enhanced by 1.2 times by the co-expression of additional tatABC from E. coli BL21(DE3). Our findings thus suggest a function of the ß-subunit of OcAOD in membrane translocation, and that the recombinant OcAOD has characteristics that are suitable for the enzymatic synthesis of glyoxylic acid as well as native OcAOD.

YnbB is a CdsA paralogue dedicated to biosynthesis of glycolipid MPIase involved in membrane protein integration.

MPIase is a glycolipid involved in protein integration in E. coli. Recently, we identified CdsA, a CDP-diacylglycerol (CDP-DAG) synthase, as a biosynthetic enzyme for MPIase. YnbB is a CdsA paralogue with a highly homologous C-terminal half. Under CdsA-depleted conditions, YnbB overproduction restored MPIase expression, but not phospholipid biosynthesis. YnbB complemented the growth defect of the cdsA knockout when Tam41p, a mitochondrial CDP-DAG synthase, was co-expressed, suggesting that YnbB possesses sufficient activity for MPIase biosynthesis, but not for phospholipid biosynthesis. Consistently, a chimera consisting of the CdsA N-terminal half and the YnbB C-terminal half (CdsA-N-YnbB-C) complemented the cdsA knockout by itself, but a chimera consisting of the YnbB N-terminal half and the CdsA C-terminal half (YnbB-N-CdsA-C) required co-expression of Tam41p for the complementation. The biosynthetic rate for CDP-DAG in CdsA and CdsA-N-YnbB-C was much faster than that in YnbB and YnbB-N-CdsA-C, indicating that the N-terminal half of CdsA accelerates CDP-DAG biosynthesis to give the fast cell growth. Therefore, the role of YnbB seems to be as a backup for MPIase biosynthesis, suggesting that YnbB is dedicated to MPIase biosynthesis. A mutant with a high pH-sensitive CdsA8 was unable to grow even under permissive conditions when the ynbB gene was deleted, supporting its auxiliary role in the CdsA function.

Syntheses and Activities of the Functional Structures of a Glycolipid Essential for Membrane Protein Integration.

MPIase is the first known glycolipid that is essential for membrane protein integration in the inner membrane of E. coli. Since the amount of natural MPIase available for analysis is limited and it contains structural heterogeneity, precisely designed synthetic derivatives are promising tools for further elucidation of its membrane protein integration mechanism. Thus, we synthesized the minimal unit of MPIase, a trisaccharyl pyrophospholipid termed mini-MPIase-3, and its derivatives. Integration assays revealed that the chemically synthesized trisaccharyl pyrophospholipid possesses significant activity, indicating that it includes the essential structure for membrane integration. Structure-activity relationship studies demonstrated that the number of trisaccharide units and the 6- O-acetyl group on N-acetylglucosamine contribute to efficient integration. Furthermore, anchoring in the membrane by a lipid moiety was essential for the integration. However, the addition of phosphorylated glycans devoid of the lipid moiety in the assay solution modulated the integration activity of MPIase embedded in liposomes, suggesting an interaction between phosphorylated glycans and substrate proteins in aqueous solutions. The prevention of protein aggregation required the 6- O-acetyl group on N-acetylglucosamine, a phosphate group at the reducing end of the glycan, and a long glycan chain. Taken together, we verified the mechanism of the initial step of the translocon-independent pathway in which a membrane protein is captured by a glycan of MPIase, which maintains its structure to be competent for integration, and then MPIase integrates it into the membrane by hydrophobic interactions with membrane lipids.