Characterization of pores formed by YaeT (Omp85) from Escherichia coli

Proteins of the Omp85 family play a major role in the biogenesis of the bacterial outer membrane, since they were shown to mediate insertion of outer membrane proteins. The Escherichia coli Omp85 homologue YaeT is essential for viability, but its exact mode of action is not yet elucidated. We could show that YaeT is composed of two distinct domains, an amino-terminal periplasmic and a carboxy-terminal membrane domain. The full length YaeT and the isolated membrane domain induce pores when reconstituted in planar lipid membranes. The pores exhibit a certain variability of conductance indicating a flexible structure, which could be an essential property of a lateral opening channel releasing proteins into the bacterial outer membrane. We could further show that the periplasmic domain proves to be essential for in vivo function of YaeT.     

YaeT (Omp85) affects the assembly of lipid-dependent and lipid-independent outer membrane proteins of Escherichia coli

The Omp85 family of proteins has been found in all Gram-negative bacteria and even several eukaryotic organisms. The previously uncharacterized Escherichia coli member of this family is YaeT. The results of this study, consistent with previous Omp85 studies, show that the yaeT gene encodes for an essential cellular function. Direct examinations of the outer membrane fraction and protein assembly revealed that cells depleted for YaeT are severely defective in the biogenesis of outer membrane proteins (OMPs). Interestingly, assemblies of the two distinct groups of OMPs that follow either SurA- and lipopolysaccharide-dependent (OmpF/C) or -independent (TolC) folding pathways were affected, suggesting that YaeT may act as a general OMP assembly factor. Depletion of cells for YaeT led to the accumulation of OMPs in the fraction enriched for periplasm, thus indicating that YaeT facilitates the insertion of soluble assembly intermediates from the periplasm to the outer membrane. Our data suggest that YaeT's role in the assembly of OMPs is not mediated through a role in lipid biogenesis, as debated for Omp85 in Neisseria, thus advocating a conserved OMP assembly function of Omp85 homologues.     

First glimpse of the crystal structure of YaeT's POTRA domains.

The Omp85/YaeT family of proteins, which are conserved from bacteria to human, catalyzes insertion and assembly of proteins in the outer membrane. The structure consists of a transmembrane beta-barrel domain and a soluble polypeptide-transport-associated (POTRA) domain. The POTRA domain is critical for substrate recognition and perhaps substrate folding, while the beta-barrel domain assists in membrane insertion. The resolution of the crystal structure of the POTRA domain of the Escherichia coli YaeT protein provides a possible molecular mechanism by which the diverse group of substrates is recognized. Knowledge gained from the crystal structure may also spur the development of a novel class of chemotherapeutic inhibitors.     

Loss of outer membrane proteins without inhibition of lipid export in an Escherichia coli YaeT mutant

Escherichia coli yaeT encodes an essential, conserved outer membrane (OM) protein that is an ortholog of Neisseria meningitidis Omp85. Conflicting data with N. meningitidis indicate that Omp85 functions either in assembly of OM proteins or in export of OM lipids. The role of YaeT in E. coli was investigated with a new temperature-sensitive mutant harboring nine amino acid substitutions. The mutant stops growing after 60 min at 44 degrees C. After 30 min at 44 degrees C, incorporation of [35S]methionine into newly synthesized OM proteins is selectively inhibited. Synthesis and export of OM phospholipids and lipopolysaccharide are not impaired. OM protein levels are low, even at 30 degrees C, and the buoyant density of the OM is correspondingly lower. By Western blotting, we show that levels of the major OM protein OmpA are lower in the mutant in whole cells, membranes, and the growth medium. SecA functions as a multicopy suppressor of the temperature-sensitive phenotype and partially restores OM proteins. Our data are consistent with a critical role for YaeT in OM protein assembly in E. coli.     

Molecular architecture and function of the Omp85 family of proteins

Omp85 is a protein found in Gram-negative bacteria where it serves to integrate proteins into the bacterial outer membrane. Members of the Omp85 family of proteins are defined by the presence of two domains: an N-terminal, periplasmic domain rich in POTRA repeats and a C-terminal beta-barrel domain embedded in the outer membrane. The widespread distribution of Omp85 family members together with their fundamental role in outer membrane assembly suggests the ancestral Omp85 arose early in the evolution of prokaryotic cells. Mitochondria, derived from an ancestral bacterial endosymbiont, also use a member of the Omp85 family to assemble proteins in their outer membranes. More distant relationships are seen between the Omp85 family and both the core proteins in two-partner secretion systems and the Toc75 family of protein translocases found in plastid outer envelopes. Aspects of the ancestry and molecular architecture of the Omp85 family of proteins is providing insight into the mechanism by which proteins might be integrated and assembled into bacterial outer membranes.     

Membrane protein insertion: mixing eukaryotic and prokaryotic concepts

Proteins are translocated across or inserted into membranes by machines that are composed of soluble and membrane-anchored subunits. The molecular action of these machines and their evolutionary origin are at present the focus of intense research. For instance, our understanding of the mode of insertion of beta-barrel membrane proteins into the outer membrane of endosymbiotically derived organelles has increased rapidly during the past few years. In particular, the identification of the Omp85/YaeT-involving pathways in Neisseria meningitidis, Escherichia coli and cyanobacteria, and homologues of Omp85/YaeT in chloroplasts and mitochondria, has provided new clues about the ancestral beta-barrel protein insertion pathway. This review focuses on recent advances in the elucidation of the evolutionarily conserved concepts that underlie the translocation and insertion of beta-barrel membrane proteins.     

Secondary structure and 1H, 13C and 15N resonance assignments of the Escherichia coli YaeT POTRA domain

We report the first ¹H, ¹³C and ¹⁵N chemical shift assignments and secondary structure of the Escherichia coli YaeT POTRA domain; a domain found in the Omp85 family of proteins which is critical for insertion and folding of outer membrane proteins in Gram-negative bacteria.     

Fold and function of polypeptide transport-associated domains responsible for delivering unfolded proteins to membranes

Membranes of Gram-negative bacteria, mitochondria and chloroplasts receive and fold beta-barrel transmembrane proteins through the action of polypeptide transport-associated (POTRA) domains. In Escherichia coli, folding substrates are inserted into the outer membrane by the essential protein YaeT, a prototypic Omp85 protein. Here, the articulation between tandem POTRA domains in solution is defined by nuclear magnetic resonance (NMR) spectroscopy, indicating an unprecedented juxtaposition. The novel solution orientations of all five POTRA domains are revealed by small-angle X-ray scattering of the entire 46 kDa periplasmic region. NMR titration studies show that strands from YaeT's canonical folding substrate, PhoE, bind non-specifically along alternating sides of its mixed beta sheets, thus providing an ideal platform for helping to fold nascent outer-membrane proteins. Together, this provides the first structural model of how multiple POTRA domains recruit substrates from the periplasmic solution into the outer membrane.     

Functioning of outer membrane protein assembly factor Omp85 requires a single POTRA domain

beta-Barrel proteins are present in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. The central component of their assembly machinery is called Omp85 in bacteria. Omp85 is predicted to consist of an integral membrane domain and an amino-terminal periplasmic extension containing five polypeptide-transport-associated (POTRA) domains. We have addressed the function of these domains by creating POTRA domain deletions in Omp85 of Neisseria meningitidis. Four POTRA domains could be deleted with only slight defects in Omp85 function. Only the most carboxy-terminal POTRA domain was essential, as was the membrane domain. Thus, similar to the mitochondrial Omp85 homologue, the functional core of bacterial Omp85 consists of its membrane domain and a single POTRA domain, that is, POTRA5.     

The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.     

Conserved Residues of the Putative L6 Loop of Escherichia coli BamA Play a Critical Role in the Assembly of β-Barrel Outer Membrane Proteins, Including That of BamA Itself

Many members of the Omp85 family of proteins form essential β-barrel outer membrane protein (OMP) biogenesis machinery in Gram-negative bacteria, chloroplasts, and mitochondria. In Escherichia coli, BamA, a member of the Omp85 family, folds into an outer membrane-embedded β-barrel domain and a soluble periplasmic polypeptide-transport-associated (POTRA) domain. Although the high-resolution structures of only the BamA POTRA domain of E. coli are available, the crystal structure of FhaC, an Omp85 family member and a component of the two-partner secretion system in Bordetella pertussis, suggests that the BamA β-barrel likely folds into a 16-stranded β-barrel. The FhaC β-barrel is occluded by an N-terminal α-helix and a large β-barrel loop, L6, which carries residues that are highly conserved among the Omp85 family members. Deletion of L6 in FhaC did not affect its biogenesis but abolished its secretion function. In this study, we tested the hypothesis that the conserved residues of the putative L6 loop, which presumably folds back into the lumen of the BamA β-barrel like the FhaC counterpart, play an important role in OMP and/or BamA biogenesis. The conserved (641)RGF(643) residues of L6 were either deleted or replaced with alanine in various permutations. Phenotypic and biochemical characterization of various BamA L6 mutants revealed that the conserved RGF residues are critical for OMP biogenesis. Moreover, three BamA L6 alterations, ΔRGF, AAA, and AGA, produced a conditional lethal phenotype, concomitant with severely reduced BamA levels and folding defects. Thus, the conserved (641)RGF(643) residues of the BamA L6 loop are important for BamA folding and biogenesis.     

Omp85 from the Thermophilic Cyanobacterium Thermosynechococcus elongatus Differs from Proteobacterial Omp85 in Structure and Domain Composition

Omp85 proteins are essential proteins located in the bacterial outer membrane. They are involved in outer membrane biogenesis and assist outer membrane protein insertion and folding by an unknown mechanism. Homologous proteins exist in eukaryotes, where they mediate outer membrane assembly in organelles of endosymbiotic origin, the mitochondria and chloroplasts. We set out to explore the homologous relationship between cyanobacteria and chloroplasts, studying the Omp85 protein from the thermophilic cyanobacterium Thermosynechococcus elongatus. Using state-of-the art sequence analysis and clustering methods, we show how this protein is more closely related to its chloroplast homologue Toc75 than to proteobacterial Omp85, a finding supported by single channel conductance measurements. We have solved the structure of the periplasmic part of the protein to 1.97 Å resolution, and we demonstrate that in contrast to Omp85 from Escherichia coli the protein has only three, not five, polypeptide transport-associated (POTRA) domains, which recognize substrates and generally interact with other proteins in bigger complexes. We model how these POTRA domains are attached to the outer membrane, based on the relationship of Omp85 to two-partner secretion system proteins, which we show and analyze. Finally, we discuss how Omp85 proteins with different numbers of POTRA domains evolved, and evolve to this day, to accomplish an increasing number of interactions with substrates and helper proteins.     

The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane

In Gram-negative bacteria, lipopolysaccharide and phospholipid biosynthesis takes place at the inner membrane. How the completed lipid molecules are subsequently transported to the outer membrane remains unknown. Omp85 of Neisseria meningitidis is representative for a family of outer membrane proteins conserved among Gram-negative bacteria. We first demonstrated that the omp85 gene is co-transcribed with genes involved in lipid biosynthesis, suggesting an involvement in lipid assembly. A meningococcal strain was constructed in which Omp85 expression could be switched on or off through a tac promoter-controlled omp85 gene. We demonstrated that the presence of Omp85 is essential for viability. Depletion of Omp85 leads to accumulation of electron-dense amorphous material and vesicular structures in the periplasm. We demonstrated, by fractionation of inner and outer membranes, that lipopolysaccharide and phospholipids mostly disappeared from the outer membrane and instead accumulated in the inner membrane, upon depletion of Omp85. Omp85 depletion did not affect localization of integral outer membrane proteins PorA and Opa. These results provide compelling evidence for a role for Omp85 in lipid transport to the outer membrane.     

Omp85(Tt) from Thermus thermophilus HB27: an ancestral type of the Omp85 protein family

Proteins belonging to the Omp85 family are involved in the assembly of beta-barrel outer membrane proteins or in the translocation of proteins across the outer membrane in bacteria, mitochondria, and chloroplasts. The cell envelope of the thermophilic bacterium Thermus thermophilus HB27 is multilayered, including an outer membrane that is not well characterized. Neither the precise lipid composition nor much about integral membrane proteins is known. The genome of HB27 encodes one Omp85-like protein, Omp85(Tt), representing an ancestral type of this family. We overexpressed Omp85(Tt) in T. thermophilus and purified it from the native outer membranes. In the presence of detergent, purified Omp85(Tt) existed mainly as a monomer, composed of two stable protease-resistant modules. Circular dichroism spectroscopy indicated predominantly beta-sheet secondary structure. Electron microscopy of negatively stained lipid-embedded Omp85(Tt) revealed ring-like structures with a central cavity of approximately 1.5 nm in diameter. Single-channel conductance recordings indicated that Omp85(Tt) forms ion channels with two different conducting states, characterized by conductances of approximately 0.4 nS and approximately 0.65 nS, respectively.     

Tic22 from Anabaena sp. PCC 7120 with holdase function involved in outer membrane protein biogenesis shuttles between plasma membrane and Omp85

β‐barrel‐shaped outer membrane proteins (OMPs) ensure regulated exchange of molecules across the cell‐wall of Gram‐negative bacteria. They are synthesized in the cytoplasm and translocated across the plasma membrane via the SEC translocon. In the periplasm, several proteins participate in the transfer of OMPs to the outer membrane‐localized complex catalyzing their insertion. This process has been described in detail for proteobacteria and some molecular components are conserved in cyanobacteria. For example, Omp85 proteins that catalyze the insertion of OMPs into the outer membrane exist in cyanobacteria as well. In turn, SurA and Skp involved in OMP transfer from plasma membrane to Omp85 in E. coli are likely replaced by Tic22 in cyanobacteria. We describe that anaTic22 functions as periplasmic holdase for OMPs in Anabaena sp. PCC 7120 and provide evidence for the process of substrate delivery to anaOmp85. AnaTic22 binds to the plasma membrane with specificity for phosphatidylglycerol and monogalactosyldiacylglycerol. Substrate recognition induces membrane dissociation and interaction with the N‐terminal POTRA domain of Omp85. This leads to substrate release by the interaction with a proline‐rich domain and the first POTRA domain of Omp85. The order of events during OMP transfer from plasma membrane to Omp85 in cyanobacteria is discussed.     

Differential effects of yfgL mutation on Escherichia coli outer membrane proteins and lipopolysaccharide

YfgL together with NlpB, YfiO, and YaeT form a protein complex to facilitate the insertion of proteins into the outer membrane of Escherichia coli. Without YfgL, the levels of OmpA, OmpF, and LamB are significantly reduced, while OmpC levels are slightly reduced. In contrast, the level of TolC significantly increases in a yfgL mutant. When cells are depleted of YaeT or YfiO, levels of all outer membrane proteins examined, including OmpC and TolC, are severely reduced. Thus, while the assembly pathways of various nonlipoprotein outer membrane proteins may vary through the step involving YfgL, all assembly pathways in Escherichia coli converge at the step involving the YaeT/YfiO complex. The negative effect of yfgL mutation on outer membrane proteins may in part be due to elevated sigma E activity, which has been shown to downregulate the synthesis of various outer membrane proteins while upregulating the synthesis of periplasmic chaperones, foldases, and lipopolysaccharide. The data presented here suggest that the yfgL effect on outer membrane proteins also stems from a defective assembly apparatus, leading to aberrant outer membrane protein assembly, except for TolC, which assembles independent of YfgL. Consistent with this view, the simultaneous absence of YfgL and the major periplasmic protease DegP confers a synthetic lethal phenotype, presumably due to the toxic accumulation of unfolded outer membrane proteins. The results support the hypothesis that TolC and major outer membrane proteins compete for the YaeT/YfiO complex, since mutations that adversely affect synthesis or assembly of major outer membrane proteins lead to elevated TolC levels.     

Molecular and functional characterisation of the Serratia marcescens outer membrane protein Omp1

Serratia marcescens outer membrane contains three different general diffusion porins: Omp1, Omp2 and Omp3. Omp1 was cloned and sequenced and it shows a great homology to the family of outer membrane porins that comprises the general porins of enteric bacteria. The gene for Omp1 was transferred into an expression plasmid and was expressed in Escherichia coli UH302 (E. coli UH302 pOM100), a porin deficient strain. Its expression confers a higher susceptibility towards different antibiotics to this strain. Omp1 was purified to homogeneity from outer membrane of E. coli UH302 pOM100. Reconstitution of the purified protein into black lipid bilayers demonstrated that it is a channel-forming component with a single-channel conductance of approximately 2 nS in 1 M KCl similar to that of other porins from enteric bacteria. Omp1 is slightly cation-selective. Its homology to already crystallised members of the family of enteric porins whose three-dimensional-structures are known and allowed the design of a topology model for Omp1. The charge distribution within a porin monomer is similar as in other general diffusion pores. The positively charged amino acids localised at the beta-strands opposite the external loop L3, which restrict the pore diameter in the porin monomer.     

Chloroplast outer envelope protein P39 in Arabidopsis thaliana belongs to the Omp85 protein family

Proteins of the Omp85 family chaperone the membrane insertion of β‐barrel‐shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear‐encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N‐terminal polypeptide transport‐associated (POTRA) domains and a C‐terminal membrane‐embedded β‐barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β‐barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75‐V, which is consistent with the phylogenetic clustering of P39 in the Toc75‐V rather than the Toc75‐III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75‐III, Toc75‐V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391–1401. © 2014 Wiley Periodicals, Inc.     

耐冷希瓦氏菌外膜蛋白的体外折叠研究

目的:旨在建立耐低温革兰氏阴性菌外膜蛋白体外折叠体系,为膜蛋白合成耐低温机制提供理论基础。方法:以包涵体的形式在大肠杆菌中过量表达了来源于耐低温希瓦氏菌的OmpA同源外膜蛋白Omp74的全蛋白质和N端跨膜结构域,纯化包涵体后,用高浓度尿素或强阴离子表面活性剂溶液溶解包涵体,以非离子表面活性剂为折叠介质,建立该外膜蛋白的体外折叠体系,同时以大肠杆菌的OmpA作为对照进行了比较研究。结果:与OmpA相比,Omp74体外折叠受温度影响较小,低浓度的阴离子表面活性剂能促Omp74的折叠,但对OmpA的折叠没有影响;C端结构域抑制Omp74在表面活性剂中的折叠;Omp74在0.5%的月桂酰基麦芽糖苷(DDM)和0.4%的十二烷基肌氨酸钠的混合溶液中能达到接近100%的折叠效率。