Glycolipozyme membrane protein integrase (MPIase): recent data

Ken-ichi Nishiyama 1  and Keiko Shimamoto 2
  • 1 Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan
  • 2 Bioorganic Research Institute, Suntory Foundation for Life Sciences, 1-1-1 Wakayamadai, Shimamoto, Mishima, Osaka 618-8503, Japan
Ken-ichi Nishiyama
  • Corresponding author
  • Email
  • Further information
  • Ken-ichi Nishiyama is a Professor at the Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University (2010–present). He received his PhD degree in Agricultural Chemistry from the University of Tokyo (1994). After the JSPS fellow (1993–1996), he worked as an assistant and Associate Professor at the Institute of Molecular and Cellular Biosciences, the University of Tokyo (1996–2009). Meanwhile, he stayed in Freiburg University as an EMBO Long Term Fellow, in collaboration with Prof. Dr Matthias Müller (2002–2004). His laboratory is focusing on the molecular mechanisms underlying protein transport inside the cells.
  • Search for other articles:
  • degruyter.comGoogle Scholar
and Keiko Shimamoto
  • Further information
  • Keiko Shimamoto studied Organic Chemistry at Osaka University and received her BS degree in 1984 and her MS degree in 1986. She joined the Suntory Institute for Bioorganic Research (present: Suntory Foundation for Life Sciences) in 1986 and received her PhD degree from Osaka University in 1991. Her research interests lie in the elucidation of mechanisms of biological active natural products based on syntheses of novel molecular probes. She was awarded the Yamazaki-Teiichi Prize in 2006 and the Chemical Society of Japan Award for Creative Work for 2013.
  • Search for other articles:
  • degruyter.comGoogle Scholar

Abstract

A novel factor for membrane protein integration, from the cytoplasmic membrane of Escherichia coli, named MPIase (membrane protein integrase), has recently been identified and characterized. MPIase was revealed to be essential for the membrane integration of a subset of membrane proteins, despite that such integration reactions have been, thus far, thought to occur spontaneously. The structure determination study revealed that MPIase is a novel glycolipid comprising a glycan chain with three N-acetylated amino sugars connected to diacylglycerol through a pyrophosphate linker. As MPIase catalyzes membrane protein integration, we propose that MPIase is a glycolipozyme on the basis of its enzyme-like function. The glycan chain exhibits a molecular chaperone-like function by directly interacting with substrate membrane proteins. Moreover, MPIase also affects the dimer structure of SecYEG, a translocon, thereby significantly stimulating preprotein translocation. The molecular mechanisms of MPIase functions will be outlined.

Introduction

All living cells express integral membrane proteins in every biomembrane. As integral membrane proteins possess transmembrane (TM) domains that are hydrophobic and water insoluble, membrane integration of such proteins proceeds coupled with translation to avoid aggregation. To achieve this cotranslational integration, many components function in a concerted manner. The integration reaction can be explained by the extended ‘signal hypothesis’ (1). SRP, a signal recognition particle, binds to a hydrophobic TM domain emerging from a ribosome and targets the SRP-nascent chain-ribosome complex to the SRP receptor (SR) on a membrane. The nascent chain is next transferred to a translocon (SecYEG in bacteria and the Sec61 complex in eukaryotic cells), followed by integration. YidC or TRAM interacts with integrating membrane proteins (Figure 1A and B). Beside the Sec-dependent pathway described above, it is known that a subset of membrane proteins of small size or a C-terminal TM is integrated independently of the SRP and Sec system because SRP only binds with a nascent chain (2–6) (Figure 1C). Recently, we clarified that a glycolipid named MPIase (membrane protein integrase) is involved in both Sec-dependent and -independent integration of membrane proteins (7–9). In this article, our recent studies on the identification and structure-function relation of MPIase will be outlined.

Figure 1
Figure 1

Mechanisms for membrane protein integration.

Protein integration into the ER membrane (A) or the cytoplasmic (inner) membrane of E. coli (B and C). The Sec-dependent (B) and -independent (C) pathways are shown. MPIase is shown as ‘M’.

Citation: Biomolecular Concepts 5, 5; 10.1515/bmc-2014-0030

Spontaneous insertion and diacylglycerol (DAG)

Membrane protein integration into inverted membrane vesicles (INV) can be easily analyzed by means of the protease protection assay (2). The membrane-integrated domain is protected by the membrane from externally added protease, giving a membrane-protected fragment (MPF) (Figure 2). In the case of MtlA (mannitol permease) with six TMs, an MPF of ∼30 kDa is generated upon membrane integration (10, 11). The in vitro system involving INV prepared from mutant strains demonstrated that MtlA is targeted to a membrane by SRP and SR, followed by integration into the membrane at the SecYEG translocon; that is, it is Sec dependent (12, 13). Efficient integration into membranes was observed, even if the SecYEG translocon was absent, when MtlA was in vitro synthesized with liposomes composed of Escherichia coli phospholipids (7). This observation is not compatible at all with the in vitro results obtained using INV prepared from mutant strains (12, 13); therefore, it must be an artifact owing to the artificial membrane system (7). Nonetheless, it should reflect membrane integration because it was stimulated by SRP/SR, and resistant against urea and high-salt extraction. Thus, MtlA was proven to be spontaneously integrated into liposomes (7), while such spontaneous integration is strictly blocked in INV (12, 13). These findings led us to the hypothesis that membrane components such as minor lipids and membrane proteins in INV inhibited the spontaneous integration. A lack of such a component in pure liposomes could be the reason why MtlA was disorderly integrated into membranes. A search for such membrane components revealed that DAG, a native membrane component of E. coli, prevents the spontaneous integration of MtlA (7). Inclusion of DAG in liposomes in several ratios as to phospholipids, corresponding to a physiological concentration in INV, completely blocked the spontaneous integration of MtlA (7). Moreover, DAG also blocked the intrinsic spontaneous integration of a subset of proteins that are known to be Sec independent. M13 procoat is a membrane protein composed of a TM domain preceded by a cleavable signal sequence (14). Pf3 coat carries a TM of N-out and C-in topology (15). These proteins contain a TM with the same topology. Membrane integration of these proteins has been extensively examined during the last 30 years (3). Mutant analysis revealed that insertion of M13 procoat and Pf3 coat proteins is independent of both Sec factors and the SRP system. In in vitro analysis, in which liposomes with protease preloaded inside were used, M13 procoat was digested as a result of membrane integration (16). Therefore, it has been widely believed that these proteins are spontaneously integrated into membranes by means of the hydrophobic interaction between TMs and lipid bilayer leaflets. When liposomes containing DAG were used for M13 procoat and Pf3 coat integration, DAG also blocked the spontaneous integration of these proteins completely (7, 17). 3L-Pf3 coat is a mutant version of Pf3 coat in which three leucine residues are inserted in the middle of the TM domain (18). Although integration of M13 procoat and Pf3 coat proteins is stimulated by the membrane potential, 3L-Pf3 coat does not require the membrane potential (18). In this sense, insertion of this protein is the most spontaneous. Even if 3L-Pf3 coat was used for the integration assay, DAG clearly blocked its spontaneous integration. These results strongly suggested that the integration of proteins, which had thus far been thought be spontaneous, requires another unknown factor(s).

Figure 2
Figure 2

The in vitro assay system for protein integration into INV or liposomes.

Membrane proteins are in vitro synthesized in the presence of INV or liposomes to allow membrane integration. After the reaction, the membrane-integrated domain is protected against the externally added proteinase K, giving MPFs (indicated by blue arrows).

Citation: Biomolecular Concepts 5, 5; 10.1515/bmc-2014-0030

The blockage of spontaneous integration of MtlA by DAG enables negative control of the reconstitution of MtlA integration. However, proteoliposomes containing SecYEG and YidC in the presence of DAG were not active in MtlA integration, again strongly suggesting the presence of a new integration factor (7).

DAG is a physiological component of E. coli membranes (19–21). The existence of DAG has been shown; however, its precise function remains unclear. It is feasible that the in vivo role of DAG is to block deregulated spontaneous insertion (7, 17). Wild-type E. coli cells express DAG at ∼1.5% as to phospholipids (17). A dgkA mutant, deficient in DAG kinase, expresses up to ∼10% DAG (19, 20). However, overproduction of dgkA did not affect the DAG expression, suggesting the occurrence of tight regulation as to DAG expression (17). Several genes are known to be involved in DAG biosynthesis, i.e., mdoB (22, 23), pgpB (24–26), and eptB (27). The products of these genes produce DAG as committed reactions. Even with the triple knockout of these genes, DAG is still expressed as in the wild type (17). Even if dgkA was overproduced in the triple knockout background, the DAG level did not change (17). These results suggest an important role of DAG, such as blockage of spontaneous insertion. Depletion of DAG will be necessary to demonstrate the precise role of DAG in E. coli.

YidC

YidC has been identified as a factor that interacts with the SecYEG translocon (28). Although inclusion of YidC with SecYEG did not affect the efficiency of translocation of presecretory proteins, the gene knockout resulted in cell death, causing defects in membrane protein integration (29). YidC is homologous to mitochondrial Oxa1p and Alb3p in chloroplasts, both of which are involved in membrane protein biogenesis (30). It is known that integration of many membrane proteins, including not only M13 procoat and Pf3 coat, but also F0c, CyoA, etc., becomes defective upon YidC depletion (31, 32). In in vitro experiments, the integrating nascent chains of membrane proteins are efficiently cross-linked to YidC, confirming that YidC interacts and functions together with SecYEG (33, 34). A membrane chaperon function has also been suggested (35). Proteoliposomes containing YidC are reported to stimulate the integration of a subset of membrane proteins, indicating that YidC is a membrane protein insertase (36).

Discovery of MPIase

It seems obvious that YidC functions as a protein insertase. Now, let us look back at 3L-Pf3 coat integration. Serek et al. (36) reported that YidC is a membrane protein insertase, but that 3L-Pf3 coat insertion occurs spontaneously irrespective of the presence and absence of YidC. In contrast, 3L-Pf3 coat insertion into liposomes was completely blocked by the presence of a physiological level of DAG (17). Moreover, in the presence of DAG, YidC-proteoliposomes were totally inactive in protein integration (7, 17, 37). Then, what is involved in the integration of these proteins if the spontaneous mechanism is not operative? Also, an attempt to reconstitute MtlA integration suggested that SecYEG, SRP, and YidC are not enough for protein integration (7). Therefore, we embarked on a search for the responsible factor in cytoplasmic membranes of E. coli.

A cholate extract of E. coli inner membranes does not contain detectable levels of Sec factors, YidC, or SRP, after it has been precipitated with acetone and then solubilized in a detergent solution (7). When this cholate extract was coreconstituted with SecYEG, MtlA integration was obviously observed in an SRP-dependent manner (7), suggesting that this extract contains the required integration factor. The cholate extract was also active in M13 procoat integration (7). The integration activity was further enhanced through several column chromatographies (7, 8). A factor-enriched fraction gave a single band corresponding to ∼7 kDa on a urea-SDS gel (7, 8). Note that this band is detected at the position of ∼12 kDa in the absence of urea (9). This fraction was active in both M13 procoat and MtlA integration (7, 17), suggesting that the same factor functions in both Sec-independent and -dependent integration. Proteinase K digestion of this fraction caused a slight shift of the band and inactivation (7). These results suggested that this factor is proteinaceous; however, it turned out that this was not the case. As described hereinafter, we reveal that the factor is a glycolipid (7, 9). This factor is characteristic in that it is soluble in trichloroacetic acid (TCA) in the presence of a detergent (7). Lipopolysaccharides (LPS), a major outer membrane component, exhibit similar TCA solubility and overlap with the factor on an SDS gel (8, 38). Moreover, mutant INV prepared from a deep rough mutant that expresses LPS with shorter saccharides (39) revealed reduced integration activity (7). From these data, we suspected that this integration factor is an LPS derivative at this stage (7). Later, it was found that this sample was slightly contaminated by LPS (8). TLC analysis involving a chromogenic reaction with anisaldehyde revealed that the factor gives a brown spot, while LPS gives a gray one (8). Further purification by liquid-liquid partition chromatography enabled separation of the factor from LPS. Thus, an LPS-free preparation was obtained. This highly pure factor exhibited high integration activity (8). Stoichiometric analysis indicated that the factor caused the integration of a higher number of 3L-Pf3 coat molecules if its molecular weight is assumed to be 7 kDa (8); that is, it catalyzed membrane integration like an enzyme. Thus, we named this factor MPIase after its function (8).

Structure and function of MPIase

Structure determination of MPIase

Amino acid analysis after HCl hydrolysis of the highly purified MPIase gave no amino acids but only two peaks corresponding to glucosamine and ammonia, suggesting that MPIase is non-proteinaceous and consists of unstable amino sugars (9). Composition analysis by gas chromatography-mass spectrometry (GC-MS) after methanolysis of MPIase also demonstrated the presence of glucosamine and fatty acids, indicating that MPIase is a glycolipid (9).

Matrix assisted laser desorption (MALDI)-time of flight (TOF)-MS of MPIase showing a characteristic repeating pattern with 608 or 650 mass intervals suggested that MPIase comprises multiple units of these sizes. The presence of many peaks at 42 mass unit intervals indicates various extents of acetylation in the molecule. MS/MS analyses of the material at m/z 608 showed that it consists of three components. One of the components was identified as N-acetyl-glucosamine (GlcNAc), and GlcNAc residues were non-stoichiometrically O-acetylated. The other building blocks were presumed to be N-acetyl-aminodeoxyhexose and N-acetyl-aminohexuronic acid, respectively, from their mass numbers (9).

Extensive nuclear magnetic resonance (NMR) investigations of MPIase supported that MPIase is composed of trisaccharide repeating units and a lipid moiety. The presence of two phosphorus atoms was demonstrated on 31P-NMR, which were assigned as pyrophosphate diester linkers between the reducing terminal of the glycan and the lipid part. The pyrophosphate linker was cleaved through mild hydrolysis with 48% aqueous HF to give a hydrophilic glycan fraction (HF-MPIase). Composition analyses by GC-MS of HF-MPIase gave three major peaks, one of which proved to be GlcNAc. The other peaks were assigned as 4-acetamido-4-deoxyfucose (Fuc4NAc) and 2-acetamido-2-deoxymannuronic acid (ManNAcA) through comparison with some synthetic authentic sugars. The sequence and linkage mode of the trisaccharide were determined by two-dimensional NMR of HF-MPIase. Moreover, we synthesized several model disaccharides and compared their 13C-NMR spectra with that of HF-MPIase. The number of repeating trisaccharide units was determined to be 9–11 by MALDI-TOF-MS after hydrolysis of MPIase with cold NaOH (NaOH-MPIase) (9).

Taking the aforementioned information into account, we determined the structure of MPIase to be as shown in Figure 3 (9).

Figure 3
Figure 3

Structure of MPIase.

Citation: Biomolecular Concepts 5, 5; 10.1515/bmc-2014-0030

Structure-activity relation

The glycan chain of MPIase is similar to that of enterobacterial common antigen (ECA), an outer membrane component (40–43), except for the length of the glycan. The glycan length of MPIase is homogeneous (n=9–11) (9), while that of ECA is longer and heterogeneous (n=18–55) (40, 41). Irrespective of the similarity in glycan structure, ECA was completely inactive in integration, indicating that the length of the glycan is critical (9). Another difference between MPIase and ECA is the linker that connects the glycan chain to DAG. Pyrophosphate is used in MPIase, whereas monophosphate is used in ECA (40, 41). This difference might reflect the subcellular localization of the two compounds. A soluble glycan chain (PP-MPIase) can be prepared by digesting MPIase with pyrophosphatase. PP-MPIase was found to be more active than the intact MPIase (9). Therefore, the difference in linker structure is not important for the function. Mild alkaline-treated MPIase (NaOH-MPIase) has a similar structure to PP-MPIase except that NaOH-MPIase lacks O-acetyl residues. NaOH-MPIase was completely inactive, indicating that the O-acetyl residues are essential for the function (9). Proteinase K-inactivation of MPIase (7) can be explained if proteinase K would digest the N-acetyl residues with its low substrate specificity (44). These results indicate that the acetyl residues of MPIase are essential for the function. When MPIase directly binds to substrate membrane proteins, the hydrophobicity provided by the acetyl residues seems to be involved in this interaction. Gel filtration analysis also suggested that MPIase forms an oligomer (9). If MPIase is assumed to form an octamer, then one MPIase complex has 250–300 acetyl residues.

Mechanism of Sec-independent integration as a membrane protein integrase

A soluble MPIase derivative, PP-MPIase, forms a soluble complex with a Sec-independent substrate of membrane proteins, indicating that MPIase possesses a molecular chaperone-like function dedicated to membrane proteins (9). The integration activity was observed even if liposomes were posttranslationally added to the PP-MPIase/membrane protein complex, indicating that formation of the complex is an intermediate step of the integration (9). Therefore, as a first step of integration, the MPIase complex receives a newborn membrane protein from a ribosome by means of its chaperone-like function. On the membrane surface, MPIase may open a putative gate into the lipid bilayer with a structure change of each subunit of MPIase, or may induce a conformational change of the protein. Through hydrophobic relay from MPIase to lipids, the substrate would be integrated into a membrane, resulting in its release from the MPIase. The MPIase free of a substrate protein gets ready for the next cycle of integration. Thus, MPIase catalyzes membrane protein integration like an enzyme, like a membrane protein integrase (Figure 4) (9). Indeed, even when a higher amount of membrane protein was synthesized than that of MPIase, MPIase drove the protein integration reaction beyond its stoichiometric amount (8). As PP-MPIase lacks the lipid anchor, it can receive a membrane protein more efficiently in a soluble milieu, explaining the higher activity of PP-MPIase (9). However, the membrane anchor of MPIase is essential for the Sec-dependent reaction (see below).

Figure 4
Figure 4

Working model for Sec-independent integration.

Newly synthesized membrane proteins are received by the glycan chains of MPIase (1). Possible structural changes of MPIase allow integration into the lipid bilayer (2–4). Upon release of a membrane protein, MPIase is recruited for the next cycle to ensure the catalytic reaction (5).

Citation: Biomolecular Concepts 5, 5; 10.1515/bmc-2014-0030

Mechanism of the Sec-dependent reaction

Identification and characterization of MPIase began with the reconstitution of MtlA integration, a Sec-dependent reaction, although a detailed analysis of the structure-function relation has been performed for a Sec-independent reaction, as summarized above. In the early analysis, we succeeded in inducing MtlA integration in an SRP-dependent manner when both SecYEG and MPIase were present (7). Therefore, it is highly likely that MPIase is involved in MtlA integration. However, it has been a matter of debate about whether MtlA integration requires MPIase, as it was reported that SecYEG or YidC is sufficient for MtlA integration (45). Nevertheless, the involvement of MPIase in preprotein translocation has been clearly demonstrated (7, 46). When MPIase was coreconstituted with SecYEG, the translocation activity of pOmpA was significantly (>10-fold) stimulated (46). One of the possible reasons for this stimulation was that MPIase affects the dimer orientation of SecYEG (46). The crystal structure of SecYEG revealed that SecYEG forms a dimer in a membrane with SecE being an interface, the so-called ‘back-to-back’ structure (47, 48). In this dimer, SecG is mapped at both distal sites. This dimer structure was confirmed by cross-linking experiments involving a mutant SecYEG in which a Cys residue had been introduced at a specified position (49). When SecYEG with Cys in TM 3 of SecE [SecYE(106C)G] was reconstituted into proteoliposomes, followed by oxidation, the SecE dimer was efficiently formed. In the case of SecYEG with Cys in TM 2 of SecG [SecYEG(60C)] (50), no SecG dimer was formed (46). However, when each SecYEG with a Cys residue was reconstituted together with MPIase, the SecG dimer but not the SecE dimer was efficiently formed, indicating that MPIase transformed the back-to-back dimer into a side-by-side dimer (Figure 5) (46). It has been reported that the SecG subunit of SecYEG, which significantly stimulates preprotein translocation (51–53), undergoes a topology inversion cycle coupled with preprotein translocation (54–57). It has also been demonstrated that this remarkable structure change of SecG is essential for the SecG function (54, 56). However, a controversial result has been reported, i.e., that a SecYEG mutant, in which SecY and SecG had been covalently cross linked, is fully functional (58). The difference in these observations originates from the experimental conditions. In the former study, INV prepared from wild-type cells were used, while in the latter, INV containing an overproduced amount of SecYEG were used. INV containing overproduced SecYEG enabled the embedding of more proteins than in the case of the wild type (59–61); however, the specific activity of SecYEG was significantly diminished in the SecYEG-overproduced INV (46). As the expression level of MPIase is not affected by SecYEG overproduction, MPIase is relatively lacking in SecYEG-overproducing membranes, giving an artificial back-to-back SecYEG structure (46). Indeed, topology inversion of SecG efficiently occurs when both SecYEG and MPIase are present in proteoliposomes, demonstrating that MPIase is essential for SecG inversion (46). In summary, SecYEG on interaction with MPIase gives a side-by-side structure in which SecG inversion becomes possible, which results in much higher preprotein translocation activity.

Figure 5
Figure 5

MPIase modulates the dimer orientation of SecYEG.

The structure of the SecYEG protomer (48) was modeled into back-to-back (left) and side-by-side (right) dimers. SecY, purple; SecE, yellow; SecG, blue. The positions of SecE 106C and SecG 60C are shown by red circles. The positions of TM 1 and 2 of SecG, and the plug domain in SecY (64) are also shown.

Citation: Biomolecular Concepts 5, 5; 10.1515/bmc-2014-0030

MPIase as a glycolipozyme

As stated in the previous chapter, MPIase has two distinct functions; one involves its protein integrase-like activity in the Sec-independent integration, and the other is its involvement in the Sec-dependent reaction. While some steps during the integration reaction are speculative, the molecular mechanisms for the catalytic cycle in the Sec-independent integration have been elucidated (Figure 4) (9). Considering that MPIase catalyzes membrane protein integration, it is plausible that MPIase functions like an enzyme that can be called a ‘glycolipozyme’ (9). In the Sec-dependent reaction, it has been demonstrated that MPIase is crucial for the activation of SecYEG through a change in structure, and acts as a protein translocase/integrase (46). In this case, MPIase functions as if it is a coenzyme or an effector of SecYEG. Nevertheless, it is still possible that MPIase functions as an integrase for the Sec-dependent integration. Overall, we conclude that MPIase plays an important role in protein integration/translocation through its enzyme-like function. Researching MPIase will not only clarify the detailed mechanisms underlying protein integration/translocation observed in all living things, but also shed new light on glycolipid biology.

Future perspectives

Analysis of the MPIase function is about to begin. Therefore, many things remain to be clarified, for example, how MPIase is biosynthesized, whether MPIase is involved in in vivo reactions, and what is the relation between MPIase and YidC.

As no biosynthetic genes for MPIase have been identified thus far, it is presently impossible to deplete MPIase of membranes in vivo to examine whether MPIase functions as a protein integrase in vivo and in vitro. The glycan structure of MPIase is similar to that of ECA; however, all the ECA-deficient mutants thus far examined expressed MPIase like wild-type cells (9). Therefore, while genes for ECA biosynthesis might be involved in MPIase biosynthesis, a separate set of genes for MPIase biosynthesis should be operative.

YidC has been extensively analyzed both in vivo and in vitro. Moreover, the recently reported crystal structure provides detailed mechanisms for membrane integration (62). However, YidC-proteoliposomes carrying a sufficient amount of DAG exhibit marginal integration activity for Sec-independent substrates (7, 17, 37). As it is clear that YidC is involved in membrane protein biogenesis, it is likely that MPIase and YidC function in a concerted manner. Nonetheless, as an in vitro integration assay relies on that a part of membrane-integrated proteins confers proteinase resistance, even partially and incompletely integrated proteins may be evaluated as being integrated. Therefore, integration may be halfway through upon the MPIase function, and then YidC may receive a substrate from MPIase, followed by completion of integration. These possibilities will be verified in the near future.

About Sec-dependent integration such as MtlA integration, a consensus view of it has not yet been formed. In our view, both SecYEG and MPIase are necessary (7); however, in others’ view, SecYEG or YidC is sufficient for MtlA integration (45). Future analysis is necessary in which spontaneous integration of MtlA is completely excluded because several detergents such as dodecylmaltoside form an insoluble complex with DAG (7, 63), which results in the depletion of DAG from liposomes that causes unexpected spontaneous integration. If MPIase is required for MtlA integration, there is another question. As MPIase affects the dimer structure of SecYEG, one could ask whether only the side-by-side dimer is active in MtlA integration or whether MPIase functions as an integrase even in the Sec-dependent integration. Subsequently, what is the role of YidC in MtlA integration, as it has been demonstrated that the nascent chain of MtlA is efficiently cross-linked with YidC (34)?

For preprotein translocation, it is believed that a preprotein is threaded through a pore in SecY, in which the opening is regulated by a plug domain (Figure 5) (64). Unlike translocation into endoplasmic reticulum (ER) membranes and in archaea where preprotein translocation proceeds cotranslationally, it is known that even a partially folded domain can be translocated posttranslationally through the SecYEG translocon in E. coli (65, 66). In this case, the pore size of SecYEG should become larger. Therefore, it is possible that the SecYEG dimer forms a larger pore with a dynamic structure change. As MPIase affects the dimer structure of SecYEG (46), MPIase may be involved in this structure change.

The molecular mechanisms suggest that every kingdom of life uses a similar system for protein integration into membranes (Figure 1A and B). Bacteria to higher plants and animals all have the SRP system and a highly homologous translocon (SecYEG and Sec61). YidC homologues can be found in mitochondria and chloroplasts (30). TRAM in the ER membrane may have a similar function to YidC in that nascent membrane proteins can be efficiently cross-linked (67). Thus, the Sec-dependent integration into the bacterial cytoplasmic membrane and the ER membrane of eukaryotic cells is rather similar (Figure 1A and B). In this sense, it is possible that MPIase homologues may be widely conserved in all living cells; eukaryotic cells including mitochondria and chloroplasts may possess a compound that functions like a membrane protein integrase similar to MPIase, even if their chemical structures may greatly differ. Future discovery of such compounds is awaited.

Acknowledgments

We thank all the people who joined the MPIase study, especially Drs. M. Maeda, M. Moser, R. Nagase, M. Huber, Y. Kawashima, S. Kusumoto, and H. Tokuda. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).

List of abbreviations
DAG

diacylglycerol

ECA

enterobacterial common antigen

INV

inverted membrane vesicles

LPS

lipopolysaccharides

MPF

membrane-protected fragment

MPIase

membrane protein integrase

PP-MPIase

pyrophosphatase-treated MPIase

SR

SRP receptor

SRP

signal recognition particle

TCA

trichloroacetic acid

TM

transmembrane

References

  • 1.

    Blobel G, Walter P, Chang CN, Goldman BM, Erickson AH, Lingappa VR. Translocation of proteins across membranes: the signal hypothesis and beyond. Symp Soc Exp Biol 1979; 33: 9–36.

  • 2.

    Koch HG, Moser M, Muller M. Signal recognition particle-dependent protein targeting, universal to all kingdoms of life. Rev Physiol Biochem Pharmacol 2003; 146: 55–94.

  • 3.

    Kuhn A. Major coat proteins of bacteriophage Pf3 and M13 as model systems for Sec-independent protein transport. FEMS Microbiol Rev 1995; 17: 185–90.

  • 4.

    Hegde RS, Keenan RJ. Tail-anchored membrane protein insertion into the endoplasmic reticulum. Nat Rev Mol Cell Biol 2011; 12: 787–98.

  • 5.

    Luirink J, von Heijne G, Houben E, de Gier JW. Biogenesis of inner membrane proteins in Escherichia coli. Annu Rev Microbiol 2005; 59: 329–55.

  • 6.

    Rapoport TA, Goder V, Heinrich SU, Matlack KE. Membrane-protein integration and the role of the translocation channel. Trends Cell Biol 2004; 14: 568–75.

  • 7.

    Nishiyama K, Ikegami A, Moser M, Schiltz E, Tokuda H, Muller M. A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations. J Biol Chem 2006; 281: 35667–76.

  • 8.

    Nishiyama K, Maeda M, Abe M, Kanamori T, Shimamoto K, Kusumoto S, Ueda T, Tokuda H. A novel complete reconstitution system for membrane integration of the simplest membrane protein. Biochem Biophys Res Commun 2010; 394: 733–6.

  • 9.

    Nishiyama K, Maeda M, Yanagisawa K, Nagase R, Komura H, Iwashita T, Yamagaki T, Kusumoto S, Tokuda H, Shimamoto K. MPIase is a glycolipozyme essential for membrane protein integration. Nat Commun 2012; 3: 1260.

  • 10.

    Sugiyama JE, Mahmoodian S, Jacobson GR. Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions. Proc Natl Acad Sci USA 1991; 88: 9603–7.

  • 11.

    Vervoort EB, Bultema JB, Schuurman-Wolters GK, Geertsma ER, Broos J, Poolman B. The first cytoplasmic loop of the mannitol permease from Escherichia coli is accessible for sulfhydryl reagents from the periplasmic side of the membrane. J Mol Biol 2005; 346: 733–43.

  • 12.

    Koch HG, Hengelage T, Neumann-Haefelin C, MacFarlane J, Hoffschulte HK, Schimz KL, Mechler B, Müller M. In vitro studies with purified components reveal signal recognition particle (SRP) and SecA/SecB as constituents of two independent protein-targeting pathways of Escherichia coli. Mol Biol Cell 1999; 10: 2163–73.

  • 13.

    Koch HG, Muller M. Dissecting the translocase and integrase functions of the Escherichia coli SecYEG translocon. J Cell Biol 2000; 150: 689–94.

  • 14.

    Mandel G, Wickner W. Translational and post-translational cleavage of M13 procoat protein: extracts of both the cytoplasmic and outer membranes of Escherichia coli contain leader peptidase activity. Proc Natl Acad Sci USA 1979; 76: 236–40.

  • 15.

    Rohrer J, Kuhn A. The function of a leader peptide in translocating charged amino acyl residues across a membrane. Science 1990; 250: 1418–21.

  • 16.

    Geller BL, Wickner W. M13 procoat inserts into liposomes in the absence of other membrane proteins. J Biol Chem 1985; 260: 13281–5.

  • 17.

    Kawashima Y, Miyazaki E, Muller M, Tokuda H, Nishiyama K. Diacylglycerol specifically blocks spontaneous integration of membrane proteins and allows detection of a factor-assisted integration. J Biol Chem 2008; 283: 24489–96.

  • 18.

    Kiefer D, Kuhn A. Hydrophobic forces drive spontaneous membrane insertion of the bacteriophage Pf3 coat protein without topological control. EMBO J 1999; 18: 6299–306.

  • 19.

    Raetz CR, Newman KF. Neutral lipid accumulation in the membranes of Escherichia coli mutants lacking diglyceride kinase. J Biol Chem 1978; 253: 3882–7.

  • 20.

    Rotering H, Raetz CR. Appearance of monoglyceride and triglyceride in the cell envelope of Escherichia coli mutants defective in diglyceride kinase. J Biol Chem 1983; 258: 8068–73.

  • 21.

    Chang YY, Kennedy EP. Pathways for the synthesis of glycerophosphatides in Escherichia coli. J Biol Chem 1967; 242: 516–9.

  • 22.

    Jackson BJ, Bohin JP, Kennedy EP. Biosynthesis of membrane-derived oligosaccharides: characterization of mdoB mutants defective in phosphoglycerol transferase I activity. J Bacteriol 1984; 160: 976–81.

  • 23.

    Fiedler W, Rotering H. Characterization of an Escherichia coli mdoB mutant strain unable to transfer sn-1-phosphoglycerol to membrane-derived oligosaccharides. J Biol Chem 1985; 260: 4799–806.

  • 24.

    Icho T, Raetz CR. Multiple genes for membrane-bound phosphatases in Escherichia coli and their action on phospholipid precursors. J Bacteriol 1983; 153: 722–30.

  • 25.

    Icho T. Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product. J Bacteriol 1988; 170: 5117–24.

  • 26.

    Funk CR, Zimniak L, Dowhan W. The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase. J Bacteriol 1992; 174: 205–13.

  • 27.

    Reynolds CM, Kalb SR, Cotter RJ, Raetz CR. A phosphoethanolamine transferase specific for the outer 3-deoxy-d-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant. J Biol Chem 2005; 280: 21202–11.

  • 28.

    Scotti PA, Urbanus ML, Brunner J, de Gier JW, von Heijne G, van der Does C, Driessen AJ, Oudega B, Luirink J. YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase. EMBO J 2000; 19: 542–9.

  • 29.

    Samuelson JC, Chen M, Jiang F, Moller I, Wiedmann M, Kuhn A, Phillips GJ, Dalbey RE. YidC mediates membrane protein insertion in bacteria. Nature 2000; 406: 637–41.

  • 30.

    Kuhn A, Stuart R, Henry R, Dalbey RE. The Alb3/Oxa1/YidC protein family: membrane-localized chaperones facilitating membrane protein insertion? Trends Cell Biol 2003; 13: 510–6.

  • 31.

    van der Laan M, Bechtluft P, Kol S, Nouwen N, Driessen AJ. F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis. J Cell Biol 2004; 165: 213–22.

  • 32.

    van der Laan M, Urbanus ML, Ten Hagen-Jongman CM, Nouwen N, Oudega B, Harms N, Driessen AJ, Luirink J. A conserved function of YidC in the biogenesis of respiratory chain complexes. Proc Natl Acad Sci USA 2003; 100: 5801–6.

  • 33.

    Urbanus ML, Scotti PA, Froderberg L, Saaf A, de Gier JW, Brunner J, Samuelson JC, Dalbey RE, Oudega B, Luirink J. Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC. EMBO Rep 2001; 2: 524–9.

  • 34.

    Beck K, Eisner G, Trescher D, Dalbey RE, Brunner J, Muller M. YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids. EMBO Rep 2001; 2: 709–14.

  • 35.

    Nagamori S, Smirnova IN, Kaback HR. Role of YidC in folding of polytopic membrane proteins. J Cell Biol 2004; 165: 53–62.

  • 36.

    Serek J, Bauer-Manz G, Struhalla G, van den Berg L, Kiefer D, Dalbey R, Kuhn A. Escherichia coli YidC is a membrane insertase for Sec-independent proteins. EMBO J 2004; 23: 294–301.

  • 37.

    Stiegler N, Dalbey RE, Kuhn A. M13 procoat protein insertion into YidC and SecYEG proteoliposomes and liposomes. J Mol Biol 2011; 406: 362–70.

  • 38.

    De Castro C, Parrilli M, Holst O, Molinaro A. Microbe-associated molecular patterns in innate immunity: extraction and chemical analysis of gram-negative bacterial lipopolysaccharides. Methods Enzymol 2010; 480: 89–115.

  • 39.

    Monner DA, Jonsson S, Boman HG. Ampicillin-resistant mutants of Escherichia coli K-12 with lipopolysaccharide alterations affecting mating ability and susceptibility to sex-specific bacteriophages. J Bacteriol 1971; 107: 420–32.

  • 40.

    Kuhn HM, Meier-Dieter U, Mayer H. ECA, the enterobacterial common antigen. FEMS Microbiol Rev 1988; 4: 195–222.

  • 41.

    Rick PD, Silver RP. Enterobacterial common antigen and capsular polysaccharides. In: Neidhardt FC, editor. Escherichia coli and Salmonella, cellular and molecular biology, 2nd ed. Washington, DC: ASM Press; 1996: 104–22.

  • 42.

    Bruix M, Jimenez-Barbero J, Cronet P. Determination by NMR spectroscopy of the structure and conformational features of the enterobacterial common antigen isolated from Escherichia coli. Carbohydr Res 1995; 273: 157–70.

  • 43.

    Basu S, Kuhn HM, Neszmelyi A, Himmelspach K, Mayer H. Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029. Eur J Biochem 1987; 162: 75–81.

  • 44.

    Ebeling W, Hennrich N, Klockow M, Metz H, Orth HD, Lang H. Proteinase K from Tritirachium album Limber. Eur J Biochem 1974; 47: 91–7.

  • 45.

    Welte T, Kudva R, Kuhn P, Sturm L, Braig D, Muller M, Warscheid B, Drepper F, Koch HG. Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle. Mol Biol Cell 2012; 23: 464–79.

  • 46.

    Moser M, Nagamori S, Huber M, Tokuda H, Nishiyama K. Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation. Proc Natl Acad Sci USA 2013; 110: 9734–9.

  • 47.

    Breyton C, Haase W, Rapoport TA, Kuhlbrandt W, Collinson I. Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 2002; 418: 662–5.

  • 48.

    Zimmer J, Nam Y, Rapoport TA. Structure of a complex of the ATPase SecA and the protein-translocation channel. Nature 2008; 455: 936–43.

  • 49.

    Kaufmann A, Manting EH, Veenendaal AK, Driessen AJ, van der Does C. Cysteine-directed cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighboring SecE. Biochemistry 1999; 38: 9115–25.

  • 50.

    Nagamori S, Nishiyama K, Tokuda H. Two SecG molecules present in a single protein translocation machinery are functional even after crosslinking. J Biochem 2000; 128: 129–37.

  • 51.

    Nishiyama K, Mizushima S, Tokuda H. A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli. EMBO J 1993; 12: 3409–15.

  • 52.

    Nishiyama K, Hanada M, Tokuda H. Disruption of the gene encoding p12 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature. EMBO J 1994; 13: 3272–7.

  • 53.

    Hanada M, Nishiyama KI, Mizushima S, Tokuda H. Reconstitution of an efficient protein translocation machinery comprising SecA and the three membrane proteins, SecY, SecE, and SecG (p12). J Biol Chem 1994; 269: 23625–31.

  • 54.

    Nishiyama K, Suzuki T, Tokuda H. Inversion of the membrane topology of SecG coupled with SecA-dependent preprotein translocation. Cell 1996; 85: 71–81.

  • 55.

    Suzuki H, Nishiyama K, Tokuda H. Coupled structure changes of SecA and SecG revealed by the synthetic lethality of the secAcsR11 and delta secG::kan double mutant. Mol Microbiol 1998; 29: 331–41.

  • 56.

    Sugai R, Takemae K, Tokuda H, Nishiyama K. Topology inversion of SecG is essential for cytosolic SecA-dependent stimulation of protein translocation. J Biol Chem 2007; 282: 29540–8.

  • 57.

    Morita K, Tokuda H, Nishiyama K. Multiple SecA molecules drive protein translocation across a single translocon with SecG inversion. J Biol Chem 2012; 287: 455–64.

  • 58.

    van der Sluis EO, van der Vries E, Berrelkamp G, Nouwen N, Driessen AJ. Topologically fixed SecG is fully functional. J Bacteriol 2006; 188: 1188–90.

  • 59.

    Douville K, Price A, Eichler J, Economou A, Wickner W. SecYEG and SecA are the stoichiometric components of preprotein translocase. J Biol Chem 1995; 270: 20106–11.

  • 60.

    van der Does C, den Blaauwen T, de Wit JG, Manting EH, Groot NA, Fekkes P, Driessen AJ. SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm. Mol Microbiol 1996; 22: 619–29.

  • 61.

    Duong F, Wickner W. Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme. EMBO J 1997; 16: 2756–68.

  • 62.

    Kumazaki K, Chiba S, Takemoto M, Furukawa A, Nishiyama K, Sugano Y, Mori T, Dohmae N, Hirata K, Nakada-Nakura Y, Maturana AD, Tanaka Y, Mori H, Sugita Y, Arisaka F, Ito K, Ishitani R, Tsukazaki T, Nureki O. Structural basis of Sec-independent membrane protein insertion by YidC. Nature 2014; 509: 516–20.

  • 63.

    Zhou C, Roberts MF. Diacylglycerol partitioning and mixing in detergent micelles: relevance to enzyme kinetics. Biochim Biophys Acta 1997; 1348: 273–86.

  • 64.

    Van den Berg B, Clemons WM Jr, Collinson I, Modis Y, Hartmann E, Harrison SC, Rapoport TA. X-ray structure of a protein-conducting channel. Nature 2004; 427: 36–44.

  • 65.

    Tani K, Tokuda H, Mizushima S. Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli. Effect of membrane energization. J Biol Chem 1990; 265: 17341–7.

  • 66.

    Kato M, Mizushima S. Translocation of conjugated presecretory proteins possessing an internal non-peptide domain into everted membrane vesicles in Escherichia coli. J Biol Chem 1993; 268: 3586–93.

  • 67.

    Gorlich D, Hartmann E, Prehn S, Rapoport TA. A protein of the endoplasmic reticulum involved early in polypeptide translocation. Nature 1992; 357: 47–52.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1.

    Blobel G, Walter P, Chang CN, Goldman BM, Erickson AH, Lingappa VR. Translocation of proteins across membranes: the signal hypothesis and beyond. Symp Soc Exp Biol 1979; 33: 9–36.

  • 2.

    Koch HG, Moser M, Muller M. Signal recognition particle-dependent protein targeting, universal to all kingdoms of life. Rev Physiol Biochem Pharmacol 2003; 146: 55–94.

  • 3.

    Kuhn A. Major coat proteins of bacteriophage Pf3 and M13 as model systems for Sec-independent protein transport. FEMS Microbiol Rev 1995; 17: 185–90.

  • 4.

    Hegde RS, Keenan RJ. Tail-anchored membrane protein insertion into the endoplasmic reticulum. Nat Rev Mol Cell Biol 2011; 12: 787–98.

  • 5.

    Luirink J, von Heijne G, Houben E, de Gier JW. Biogenesis of inner membrane proteins in Escherichia coli. Annu Rev Microbiol 2005; 59: 329–55.

  • 6.

    Rapoport TA, Goder V, Heinrich SU, Matlack KE. Membrane-protein integration and the role of the translocation channel. Trends Cell Biol 2004; 14: 568–75.

  • 7.

    Nishiyama K, Ikegami A, Moser M, Schiltz E, Tokuda H, Muller M. A derivative of lipid A is involved in signal recognition particle/SecYEG-dependent and -independent membrane integrations. J Biol Chem 2006; 281: 35667–76.

  • 8.

    Nishiyama K, Maeda M, Abe M, Kanamori T, Shimamoto K, Kusumoto S, Ueda T, Tokuda H. A novel complete reconstitution system for membrane integration of the simplest membrane protein. Biochem Biophys Res Commun 2010; 394: 733–6.

  • 9.

    Nishiyama K, Maeda M, Yanagisawa K, Nagase R, Komura H, Iwashita T, Yamagaki T, Kusumoto S, Tokuda H, Shimamoto K. MPIase is a glycolipozyme essential for membrane protein integration. Nat Commun 2012; 3: 1260.

  • 10.

    Sugiyama JE, Mahmoodian S, Jacobson GR. Membrane topology analysis of Escherichia coli mannitol permease by using a nested-deletion method to create mtlA-phoA fusions. Proc Natl Acad Sci USA 1991; 88: 9603–7.

  • 11.

    Vervoort EB, Bultema JB, Schuurman-Wolters GK, Geertsma ER, Broos J, Poolman B. The first cytoplasmic loop of the mannitol permease from Escherichia coli is accessible for sulfhydryl reagents from the periplasmic side of the membrane. J Mol Biol 2005; 346: 733–43.

  • 12.

    Koch HG, Hengelage T, Neumann-Haefelin C, MacFarlane J, Hoffschulte HK, Schimz KL, Mechler B, Müller M. In vitro studies with purified components reveal signal recognition particle (SRP) and SecA/SecB as constituents of two independent protein-targeting pathways of Escherichia coli. Mol Biol Cell 1999; 10: 2163–73.

  • 13.

    Koch HG, Muller M. Dissecting the translocase and integrase functions of the Escherichia coli SecYEG translocon. J Cell Biol 2000; 150: 689–94.

  • 14.

    Mandel G, Wickner W. Translational and post-translational cleavage of M13 procoat protein: extracts of both the cytoplasmic and outer membranes of Escherichia coli contain leader peptidase activity. Proc Natl Acad Sci USA 1979; 76: 236–40.

  • 15.

    Rohrer J, Kuhn A. The function of a leader peptide in translocating charged amino acyl residues across a membrane. Science 1990; 250: 1418–21.

  • 16.

    Geller BL, Wickner W. M13 procoat inserts into liposomes in the absence of other membrane proteins. J Biol Chem 1985; 260: 13281–5.

  • 17.

    Kawashima Y, Miyazaki E, Muller M, Tokuda H, Nishiyama K. Diacylglycerol specifically blocks spontaneous integration of membrane proteins and allows detection of a factor-assisted integration. J Biol Chem 2008; 283: 24489–96.

  • 18.

    Kiefer D, Kuhn A. Hydrophobic forces drive spontaneous membrane insertion of the bacteriophage Pf3 coat protein without topological control. EMBO J 1999; 18: 6299–306.

  • 19.

    Raetz CR, Newman KF. Neutral lipid accumulation in the membranes of Escherichia coli mutants lacking diglyceride kinase. J Biol Chem 1978; 253: 3882–7.

  • 20.

    Rotering H, Raetz CR. Appearance of monoglyceride and triglyceride in the cell envelope of Escherichia coli mutants defective in diglyceride kinase. J Biol Chem 1983; 258: 8068–73.

  • 21.

    Chang YY, Kennedy EP. Pathways for the synthesis of glycerophosphatides in Escherichia coli. J Biol Chem 1967; 242: 516–9.

  • 22.

    Jackson BJ, Bohin JP, Kennedy EP. Biosynthesis of membrane-derived oligosaccharides: characterization of mdoB mutants defective in phosphoglycerol transferase I activity. J Bacteriol 1984; 160: 976–81.

  • 23.

    Fiedler W, Rotering H. Characterization of an Escherichia coli mdoB mutant strain unable to transfer sn-1-phosphoglycerol to membrane-derived oligosaccharides. J Biol Chem 1985; 260: 4799–806.

  • 24.

    Icho T, Raetz CR. Multiple genes for membrane-bound phosphatases in Escherichia coli and their action on phospholipid precursors. J Bacteriol 1983; 153: 722–30.

  • 25.

    Icho T. Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product. J Bacteriol 1988; 170: 5117–24.

  • 26.

    Funk CR, Zimniak L, Dowhan W. The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase. J Bacteriol 1992; 174: 205–13.

  • 27.

    Reynolds CM, Kalb SR, Cotter RJ, Raetz CR. A phosphoethanolamine transferase specific for the outer 3-deoxy-d-manno-octulosonic acid residue of Escherichia coli lipopolysaccharide. Identification of the eptB gene and Ca2+ hypersensitivity of an eptB deletion mutant. J Biol Chem 2005; 280: 21202–11.

  • 28.

    Scotti PA, Urbanus ML, Brunner J, de Gier JW, von Heijne G, van der Does C, Driessen AJ, Oudega B, Luirink J. YidC, the Escherichia coli homologue of mitochondrial Oxa1p, is a component of the Sec translocase. EMBO J 2000; 19: 542–9.

  • 29.

    Samuelson JC, Chen M, Jiang F, Moller I, Wiedmann M, Kuhn A, Phillips GJ, Dalbey RE. YidC mediates membrane protein insertion in bacteria. Nature 2000; 406: 637–41.

  • 30.

    Kuhn A, Stuart R, Henry R, Dalbey RE. The Alb3/Oxa1/YidC protein family: membrane-localized chaperones facilitating membrane protein insertion? Trends Cell Biol 2003; 13: 510–6.

  • 31.

    van der Laan M, Bechtluft P, Kol S, Nouwen N, Driessen AJ. F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis. J Cell Biol 2004; 165: 213–22.

  • 32.

    van der Laan M, Urbanus ML, Ten Hagen-Jongman CM, Nouwen N, Oudega B, Harms N, Driessen AJ, Luirink J. A conserved function of YidC in the biogenesis of respiratory chain complexes. Proc Natl Acad Sci USA 2003; 100: 5801–6.

  • 33.

    Urbanus ML, Scotti PA, Froderberg L, Saaf A, de Gier JW, Brunner J, Samuelson JC, Dalbey RE, Oudega B, Luirink J. Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC. EMBO Rep 2001; 2: 524–9.

  • 34.

    Beck K, Eisner G, Trescher D, Dalbey RE, Brunner J, Muller M. YidC, an assembly site for polytopic Escherichia coli membrane proteins located in immediate proximity to the SecYE translocon and lipids. EMBO Rep 2001; 2: 709–14.

  • 35.

    Nagamori S, Smirnova IN, Kaback HR. Role of YidC in folding of polytopic membrane proteins. J Cell Biol 2004; 165: 53–62.

  • 36.

    Serek J, Bauer-Manz G, Struhalla G, van den Berg L, Kiefer D, Dalbey R, Kuhn A. Escherichia coli YidC is a membrane insertase for Sec-independent proteins. EMBO J 2004; 23: 294–301.

  • 37.

    Stiegler N, Dalbey RE, Kuhn A. M13 procoat protein insertion into YidC and SecYEG proteoliposomes and liposomes. J Mol Biol 2011; 406: 362–70.

  • 38.

    De Castro C, Parrilli M, Holst O, Molinaro A. Microbe-associated molecular patterns in innate immunity: extraction and chemical analysis of gram-negative bacterial lipopolysaccharides. Methods Enzymol 2010; 480: 89–115.

  • 39.

    Monner DA, Jonsson S, Boman HG. Ampicillin-resistant mutants of Escherichia coli K-12 with lipopolysaccharide alterations affecting mating ability and susceptibility to sex-specific bacteriophages. J Bacteriol 1971; 107: 420–32.

  • 40.

    Kuhn HM, Meier-Dieter U, Mayer H. ECA, the enterobacterial common antigen. FEMS Microbiol Rev 1988; 4: 195–222.

  • 41.

    Rick PD, Silver RP. Enterobacterial common antigen and capsular polysaccharides. In: Neidhardt FC, editor. Escherichia coli and Salmonella, cellular and molecular biology, 2nd ed. Washington, DC: ASM Press; 1996: 104–22.

  • 42.

    Bruix M, Jimenez-Barbero J, Cronet P. Determination by NMR spectroscopy of the structure and conformational features of the enterobacterial common antigen isolated from Escherichia coli. Carbohydr Res 1995; 273: 157–70.

  • 43.

    Basu S, Kuhn HM, Neszmelyi A, Himmelspach K, Mayer H. Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029. Eur J Biochem 1987; 162: 75–81.

  • 44.

    Ebeling W, Hennrich N, Klockow M, Metz H, Orth HD, Lang H. Proteinase K from Tritirachium album Limber. Eur J Biochem 1974; 47: 91–7.

  • 45.

    Welte T, Kudva R, Kuhn P, Sturm L, Braig D, Muller M, Warscheid B, Drepper F, Koch HG. Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle. Mol Biol Cell 2012; 23: 464–79.

  • 46.

    Moser M, Nagamori S, Huber M, Tokuda H, Nishiyama K. Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation. Proc Natl Acad Sci USA 2013; 110: 9734–9.

  • 47.

    Breyton C, Haase W, Rapoport TA, Kuhlbrandt W, Collinson I. Three-dimensional structure of the bacterial protein-translocation complex SecYEG. Nature 2002; 418: 662–5.

  • 48.

    Zimmer J, Nam Y, Rapoport TA. Structure of a complex of the ATPase SecA and the protein-translocation channel. Nature 2008; 455: 936–43.

  • 49.

    Kaufmann A, Manting EH, Veenendaal AK, Driessen AJ, van der Does C. Cysteine-directed cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighboring SecE. Biochemistry 1999; 38: 9115–25.

  • 50.

    Nagamori S, Nishiyama K, Tokuda H. Two SecG molecules present in a single protein translocation machinery are functional even after crosslinking. J Biochem 2000; 128: 129–37.

  • 51.

    Nishiyama K, Mizushima S, Tokuda H. A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli. EMBO J 1993; 12: 3409–15.

  • 52.

    Nishiyama K, Hanada M, Tokuda H. Disruption of the gene encoding p12 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature. EMBO J 1994; 13: 3272–7.

  • 53.

    Hanada M, Nishiyama KI, Mizushima S, Tokuda H. Reconstitution of an efficient protein translocation machinery comprising SecA and the three membrane proteins, SecY, SecE, and SecG (p12). J Biol Chem 1994; 269: 23625–31.

  • 54.

    Nishiyama K, Suzuki T, Tokuda H. Inversion of the membrane topology of SecG coupled with SecA-dependent preprotein translocation. Cell 1996; 85: 71–81.

  • 55.

    Suzuki H, Nishiyama K, Tokuda H. Coupled structure changes of SecA and SecG revealed by the synthetic lethality of the secAcsR11 and delta secG::kan double mutant. Mol Microbiol 1998; 29: 331–41.

  • 56.

    Sugai R, Takemae K, Tokuda H, Nishiyama K. Topology inversion of SecG is essential for cytosolic SecA-dependent stimulation of protein translocation. J Biol Chem 2007; 282: 29540–8.

  • 57.

    Morita K, Tokuda H, Nishiyama K. Multiple SecA molecules drive protein translocation across a single translocon with SecG inversion. J Biol Chem 2012; 287: 455–64.

  • 58.

    van der Sluis EO, van der Vries E, Berrelkamp G, Nouwen N, Driessen AJ. Topologically fixed SecG is fully functional. J Bacteriol 2006; 188: 1188–90.

  • 59.

    Douville K, Price A, Eichler J, Economou A, Wickner W. SecYEG and SecA are the stoichiometric components of preprotein translocase. J Biol Chem 1995; 270: 20106–11.

  • 60.

    van der Does C, den Blaauwen T, de Wit JG, Manting EH, Groot NA, Fekkes P, Driessen AJ. SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm. Mol Microbiol 1996; 22: 619–29.

  • 61.

    Duong F, Wickner W. Distinct catalytic roles of the SecYE, SecG and SecDFyajC subunits of preprotein translocase holoenzyme. EMBO J 1997; 16: 2756–68.

  • 62.

    Kumazaki K, Chiba S, Takemoto M, Furukawa A, Nishiyama K, Sugano Y, Mori T, Dohmae N, Hirata K, Nakada-Nakura Y, Maturana AD, Tanaka Y, Mori H, Sugita Y, Arisaka F, Ito K, Ishitani R, Tsukazaki T, Nureki O. Structural basis of Sec-independent membrane protein insertion by YidC. Nature 2014; 509: 516–20.

  • 63.

    Zhou C, Roberts MF. Diacylglycerol partitioning and mixing in detergent micelles: relevance to enzyme kinetics. Biochim Biophys Acta 1997; 1348: 273–86.

  • 64.

    Van den Berg B, Clemons WM Jr, Collinson I, Modis Y, Hartmann E, Harrison SC, Rapoport TA. X-ray structure of a protein-conducting channel. Nature 2004; 427: 36–44.

  • 65.

    Tani K, Tokuda H, Mizushima S. Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli. Effect of membrane energization. J Biol Chem 1990; 265: 17341–7.

  • 66.

    Kato M, Mizushima S. Translocation of conjugated presecretory proteins possessing an internal non-peptide domain into everted membrane vesicles in Escherichia coli. J Biol Chem 1993; 268: 3586–93.

  • 67.

    Gorlich D, Hartmann E, Prehn S, Rapoport TA. A protein of the endoplasmic reticulum involved early in polypeptide translocation. Nature 1992; 357: 47–52.

OPEN ACCESS

Journal + Issues

BioMolecular Concepts is a peer-reviewed journal fostering the integration of different fields of biomolecular research. The journal provides expert summaries and conclusive extensions of research data leading to new and original, testable hypotheses. Aspects of research that can promote related fields and lead to novel insight into biological mechanisms are of special interest.

Search

  • View in gallery
  • View in gallery
  • View in gallery

    Mechanisms for membrane protein integration.

    Protein integration into the ER membrane (A) or the cytoplasmic (inner) membrane of E. coli (B and C). The Sec-dependent (B) and -independent (C) pathways are shown. MPIase is shown as ‘M’.

  • View in gallery

    The in vitro assay system for protein integration into INV or liposomes.

    Membrane proteins are in vitro synthesized in the presence of INV or liposomes to allow membrane integration. After the reaction, the membrane-integrated domain is protected against the externally added proteinase K, giving MPFs (indicated by blue arrows).

  • View in gallery

    Structure of MPIase.

  • View in gallery

    Working model for Sec-independent integration.

    Newly synthesized membrane proteins are received by the glycan chains of MPIase (1). Possible structural changes of MPIase allow integration into the lipid bilayer (2–4). Upon release of a membrane protein, MPIase is recruited for the next cycle to ensure the catalytic reaction (5).

  • View in gallery

    MPIase modulates the dimer orientation of SecYEG.

    The structure of the SecYEG protomer (48) was modeled into back-to-back (left) and side-by-side (right) dimers. SecY, purple; SecE, yellow; SecG, blue. The positions of SecE 106C and SecG 60C are shown by red circles. The positions of TM 1 and 2 of SecG, and the plug domain in SecY (64) are also shown.