The bacterial outer membrane β-barrel assembly machinery

β-Barrel proteins found in the outer membrane of Gram-negative bacteria serve a variety of cellular functions. Proper folding and assembly of these proteins are essential for the viability of bacteria and can also play an important role in virulence. The β-barrel assembly machinery (BAM) complex, which is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, has been the focus of many recent studies. This review summarizes the significant progress that has been made toward understanding the structure and function of the bacterial BAM complex.     

Assembly of β-barrel proteins into bacterial outer membranes

Membrane proteins with a β-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of β-barrel protein assembly into membranes, and the components of the β-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein

Definitive identification of Treponema pallidum rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in T. pallidum with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modelling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic β-barrel. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the β-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in T. pallidum larger than that of Escherichia coli. Non-orthologous ancillary factors and self-association of TP0326 via its β-barrel may both contribute to the Bam complex. T. pallidum-infected rabbits mount a vigorous antibody response to both POTRA and β-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochaete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.     

Biogenesis of β-barrel integral proteins of bacterial outer membrane

Gram-negative bacteria are enveloped by two membranes, the inner (cytoplasmic) (CM) and the outer (OM). The majority of integral outer membrane proteins are arranged in β-barrels of cylindrical shape composed of amphipathic antiparallel β-strands. In bacteria, β-barrel proteins function as water-filled pores, active transporters, enzymes, receptors, and structural proteins. Proteins of bacterial OM are synthesized in the cytoplasm as unfolded polypeptides with an N-terminal sequence that marks them for transport across the CM. Precursors of membrane proteins move through the aqueous medium of the cytosol and periplasm under the protection of chaperones (SecB, Skp, SurA, and DegP), then cross the CM via the Sec system composed of a polypeptide-conducting channel (SecYEG) and ATPase (SecA), the latter providing the energy for the translocation of the pre-protein. Pre-protein folding and incorporation in the OM require the participation of the Bam-complex, probably without the use of energy. This review summarizes current data on the biogenesis of the β-barrel proteins of bacterial OM. Data on the structure of the proteins included in the multicomponent system for delivery of the OM proteins to their destination in the cell and on their complexes with partners, including pre-proteins, are pre-sented. Molecular models constructed on the basis of structural, genetic, and biochemical studies that describe the mechanisms of β-barrel protein assembly by this molecular transport machinery are also considered.     

Crystal structure of β-barrel assembly machinery BamCD protein complex

The β-barrel assembly machinery (BAM) complex of Escherichia coli is a multiprotein machine that catalyzes the essential process of assembling outer membrane proteins. The BAM complex consists of five proteins: one membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Here, we report the first crystal structure of a Bam lipoprotein complex: the essential lipoprotein BamD in complex with the N-terminal half of BamC (BamC(UN) (Asp(28)-Ala(217)), a 73-residue-long unstructured region followed by the N-terminal domain). The BamCD complex is stabilized predominantly by various hydrogen bonds and salt bridges formed between BamD and the N-terminal unstructured region of BamC. Sequence and molecular surface analyses revealed that many of the conserved residues in both proteins are found at the BamC-BamD interface. A series of truncation mutagenesis and analytical gel filtration chromatography experiments confirmed that the unstructured region of BamC is essential for stabilizing the BamCD complex structure. The unstructured N terminus of BamC interacts with the proposed substrate-binding pocket of BamD, suggesting that this region of BamC may play a regulatory role in outer membrane protein biogenesis.     

Structural biology of membrane-intrinsic β-barrel enzymes: Sentinels of the bacterial outer membrane

The outer membranes of Gram-negative bacteria are replete with integral membrane proteins that exhibit antiparallel β-barrel structures, but very few of these proteins function as enzymes. In Escherichia coli, only three β-barrel enzymes are known to exist in the outer membrane; these are the phospholipase OMPLA, the protease OmpT, and the phospholipid∷lipid A palmitoyltransferase PagP, all of which have been characterized at the structural level. Structural details have also emerged for the outer membrane β-barrel enzyme PagL, a lipid A 3-O-deacylase from Pseudomonas aeruginosa. Lipid A can be further modified in the outer membrane by two β-barrel enzymes of unknown structure; namely, the Salmonella enterica 3′-acyloxyacyl hydrolase LpxR, and the Rhizobium leguminosarum oxidase LpxQ, which employs O₂ to convert the proximal glucosamine unit of lipid A into 2-aminogluconate. Structural biology now indicates how β-barrel enzymes can function as sentinels that remain dormant when the outer membrane permeability barrier is intact. Host immune defenses and antibiotics that perturb this barrier can directly trigger β-barrel enzymes in the outer membrane. The ensuing adaptive responses occur instantaneously and rapidly outpace other signal transduction mechanisms that similarly function to restore the outer membrane permeability barrier.     

Structure and function of BamE within the outer membrane and the β-barrel assembly machine

Insertion of folded proteins into the outer membrane of Gram-negative bacteria is mediated by the essential β-barrel assembly machine (Bam). Here, we report the native structure and mechanism of a core component of this complex, BamE, and show that it is exclusively monomeric in its native environment of the periplasm, but is able to adopt a distinct dimeric conformation in the cytoplasm. BamE is shown to bind specifically to phosphatidylglycerol, and comprehensive mutagenesis and interaction studies have mapped key determinants for complex binding, outer membrane integrity and cell viability, as well as revealing the role of BamE within the Bam complex.     

Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane

The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related "TpsA" proteins across the outer membrane via their dedicated "TpsB" transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.     

Targeting of Neisserial PorB to the mitochondrial outer membrane: an insight on the evolution of β-barrel protein assembly machines

Mitochondria originated from Gram-negative bacteria through endosymbiosis. In modern day mitochondria, the Sorting and Assembly Machinery (SAM) is responsible for eukaryotic β-barrel protein assembly in the mitochondrial outer membrane. The SAM is the functional equivalent of the β-barrel assembly machinery found in the outer membrane of Gram-negative bacteria. In this study we examined the import pathway of a pathogenic bacterial protein, PorB, which is targeted from pathogenic Neisseria to the host mitochondria. We have developed a new method for measurement of PorB assembly into mitochondria that relies on the mobility shift exhibited by bacterial β-barrel proteins once folded and separated under semi-native electrophoretic conditions. We show that PorB is targeted to the outer mitochondrial membrane with a dependence on the intermembrane space shuttling chaperones and the core component of the SAM, Sam50, which is a functional homologue of BamA that is required for PorB assembly in bacteria. The peripheral subunits of the SAM, Sam35 and Sam37, which are essential for eukaryotic β-barrel protein assembly but do not have distinguishable functional homologues in bacteria, are not required for PorB assembly in eukaryotes. This shows that PorB uses an evolutionary conserved 'bacterial like' mechanism to infiltrate the host mitochondrial outer membrane.     

BamE structure: the assembly of β-barrel proteins in the outer membranes of bacteria and mitochondria

A study recently published in EMBO reports solves the solution structure of E. coli BamE, thus providing the basis for a better understanding of the mechanism of β-barrel assembly in bacterial and mitochondrial outer membranes.EMBO Rep (2011) advance online publication. doi: 10.1038/embor.2010.202β-barrel membrane proteins are found exclusively in the outer membrane of Gram-negative bacteria and the outer membranes of eukaryotic organelles of prokaryotic origin, mitochondria and chloroplasts. In contrast to the inner membrane, the bacterial outer membrane is an asymmetrical bilayer that consists mainly of lipopolysaccharides in the outer leaflet and phospholipids in the inner leaflet. Bacterial β-barrel outer membrane proteins (OMPs) mediate many cellular functions, for example, passive or selective diffusion of small molecules through the β-barrel pores across the outer membrane. By contrast, only a few mitochondrial β-barrel outer membrane proteins (MBOMPs) have been identified so far. The central machineries that mediate insertion and assembly of OMPs/MBOMPs are the β-barrel assembly machine (BAM) complex in the bacterial outer membrane and the topogenesis of outer-membrane β-barrel proteins (TOB)/sorting and assembly machinery (SAM) complex in the mitochondrial outer membrane (Knowles et al, 2009; Endo & Yamano, 2010; Stroud et al, 2010; Fig 1). However, the molecular mechanisms of β-barrel protein topogenesis in bacterial and mitochondrial outer membranes remain poorly understood.Open in a separate windowFigure 1β-barrel protein assembly in bacterial and mitochondrial outer membranes. (A) Bacteria. Ribbon models of the structures of the Sec complex, SurA, BamA (Clantin et al, 2007; Kim et al, 2007), BamE and OMP. The upper and lower inserts show the surface of BamE (residues 20–108; viewed after approximately 90° rotation of the ribbon model around the horizontal axis toward the reader). Residues important for BamD binding are shown in red and residues with NMR signals that were perturbed by BamD binding are shown in yellow. The residue (Phe 74) important for PG binding is shown in red and the residues with NMR signals that were perturbed by PG binding are shown in yellow. (B) Mitochondria. Ribbon models were drawn for the structures of small Tim and MBOMP. IM, inner membrane; IMS, intermembrane space; MBOMP, mitochondrial β-barrel outer membrane protein; OM, outer membrane; OMP, outer membrane protein; PG, phosphatidylglycerol; POTRA, polypeptide transport-associated domain.Bacterial OMPs are synthesized in the cytosol as precursor proteins with an amino-terminal signal sequence that guides the proteins to the Sec machinery for crossing the inner membrane and is cleaved off in the periplasm. Periplasmic chaperones then escort OMPs through the aqueous periplasmic space in a partly unfolded state. On reaching the outer membrane, OMPs assemble into a β-barrel structure and insert into the outer membrane with the help of the BAM complex. The bacterial OMP insertion pathway can be compared to the assembly pathway of MBOMPs from the mitochondrial intermembrane space into the outer membrane. MBOMPs are synthesized in the cytosol and imported into the intermembrane space by the outer membrane translocator TOM40. The subsequent chaperone-mediated escort across the intermembrane space and insertion into the outer membrane by the TOB complex is similar to the OMP assembly process. Notably, the BAM and TOB complexes share the homologous β-barrel proteins BamA and Tob55/Sam50, respectively, as the central components of their insertion machineries. The BAM complex in Escherichia coli consists of BamA (YaeT/Omp85) and four accessory lipoproteins: BamB (YfgL), BamC (NlpB), BamD (YfiO) and BamE (SmpA). BamA and BamD are essential for cell growth, yet deletion of dispensable BamB, BamC or BamE leads to outer membrane defects manifested in hypersensitivity to antibiotics. Although BamAB and BamCDE can form distinct subcomplexes, they become functional only after formation of the entire BAM complex with all five subunits (Hagan et al, 2010).In this issue of EMBO reports, Knowles et al (2011) solve the nuclear magnetic resonance (NMR) solution structure of E. coli BamE, which sheds light on the roles of one of the Bam subunits in β-barrel protein assembly. The structure of BamE consists of a three-stranded antiparallel β-sheet packed against a pair of α-helices (Fig 1).As the ΔbamE mutant cannot grow in the presence of vancomycin, the authors identify functionally important residues of BamE by testing the effects of amino-acid substitutions in BamE on its inability to complement the growth defects of ΔbamE, without destabilizing BamE itself. Many of the identified residues are conserved among BamE proteins from different organisms and map to a single surface area on BamE. Interestingly, NMR signals of the residues around this region are sensitive to the addition of micelles containing the lipid phosphatidylglycerol, but not phosphatidylethanolamine or cardiolipin. In parallel, the authors analyse perturbation of the NMR spectra of BamE after the addition of purified BamB, C and D proteins. Only BamD affects the NMR spectra of BamE, and the BamD interacting region of BamE is found to overlap partly with the residues involved in phosphatidylglycerol binding. As the addition of BamD and phosphatidylglycerol have different effects on the NMR spectra of BamE, the binding of BamD and phosphatidylglycerol to BamE seem to take place simultaneously. What is the biological relevance of the observed interactions of BamE with both BamD and phosphatidylglycerol? As phosphatidylglycerol was found to help the insertion of OMPs into lipid liposomes (Patel et al, 2009), BamE might recruit the BAM complex through BamD to phosphatidylglycerol-rich regions in the outer membrane, or might directly recruit phosphatidylglycerol to form assembly points for OMP insertion and folding.What are the roles of other subunits of the BAM complex in β-barrel protein assembly? The essential subunit of the E. coli BAM complex BamA consists of two domains: the N-terminal polypeptide transport-associated (POTRA) domain repeat in the periplasm and the carboxy-terminal β-barrel domain, embedded in the outer membrane. The number of POTRA domains ranges from one to five in BamA homologues from different organisms. Of these POTRA domains, the one nearest to the C-terminal that is most connected to the β-barrel domain is essential for cell viability and its deletion leads to disassembly of the BAM complex (Kim et al, 2007). Structural studies of the E. coli BamA POTRA domains suggest that each POTRA domain has a common fold, whereas conformational rigidity might differ between inter-domain linkers (Gatzeva-Topalova et al, 2010; Fig 1). As individual POTRA domains have some affinity for unfolded substrate proteins, the periplasmic tandem POTRA repeat probably provides several substrate binding sites that slide the substrate progressively towards the BamA β-barrel domain. The β-barrel domain of BamA probably functions as a scaffold to facilitate the formation of β-strands, possibly through β-augmentation and subsequent spontaneous membrane insertion of the β-barrel. Yet, it is not clear whether this cradle for β-strand formation is provided by the pore formed within the monomer or oligomeric forms of the BamA β-barrel domain. Alternatively, membrane insertion and folding of OMPs might take place at the interface between BamA and the outer membrane lipid bilayer.How much of the β-barrel assembly process is conserved during the evolution of mitochondria from Gram-negative bacteria? Although the central subunits BamA and Tob55 of the BAM and TOB complexes are conserved, other subunits of these complexes are unrelated to each other. The POTRA domains of BamA are essential for recognition and assembly of bacterial OMPs, whereas that of Tob55 is dispensable for MBOMP assembly in the mitochondrial outer membrane. Nevertheless, the mitochondrial TOB complex facilitates assembly of bacterial OMPs at low efficiency (Walther et al, 2009) and, in turn, the bacterial BAM complex can mediate assembly of mitochondrial porin. Therefore, the basic mechanism of β-barrel assembly in the outer membranes of bacteria and mitochondria seems to be conserved. High-resolution structures of each component of the BAM and TOB complexes—including that of BamE in this study—will thus provide the basis for a better understanding of the mechanism of β-barrel assembly in evolutionarily related bacterial and mitochondrial outer membranes.     

Physical determinants of β-barrel membrane protein folding in lipid vesicles

The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)(60) and Opa(50) into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.     

Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane

The TOB–SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55–Sam50, Tob38–Sam35, and Tob37–Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB–SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.     

Evaluation of methods for predicting the topology of β-barrel outer membrane proteins and a consensus prediction method

Background  

Prediction of the transmembrane strands and topology of β-barrel outer membrane proteins is of interest in current bioinformatics research. Several methods have been applied so far for this task, utilizing different algorithmic techniques and a number of freely available predictors exist. The methods can be grossly divided to those based on Hidden Markov Models (HMMs), on Neural Networks (NNs) and on Support Vector Machines (SVMs). In this work, we compare the different available methods for topology prediction of β-barrel outer membrane proteins. We evaluate their performance on a non-redundant dataset of 20 β-barrel outer membrane proteins of gram-negative bacteria, with structures known at atomic resolution. Also, we describe, for the first time, an effective way to combine the individual predictors, at will, to a single consensus prediction method.     

Dual energy landscape: the functional state of the β-barrel outer membrane protein G molds its unfolding energy landscape

We applied dynamic single-molecule force spectroscopy to quantify the parameters (free energy of activation and distance of the transition state from the folded state) characterizing the energy barriers in the unfolding energy landscape of the outer membrane protein G (OmpG) from Escherichia coli. The pH-dependent functional switching of OmpG directs the protein along different regions on the unfolding energy landscape. The two functional states of OmpG take the same unfolding pathway during the sequential unfolding of β-hairpins I-IV. After the initial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of β-hairpin V in one step precedes the unfolding of β-hairpin VI. In the closed state, β-hairpin V and β-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequently β-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the open and closed states join again in the last unfolding step of β-hairpin VII. Also, the conformational change from the open to the closed state witnesses a rigidified extracellular gating loop L6. Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways but also tunes its mechanical properties for optimum function.     

Crystal structure of Escherichia coli BamB, a lipoprotein component of the β-barrel assembly machinery complex

In Gram-negative bacteria, the BAM (β-barrel assembly machinery) complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one β-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their role in outer membrane protein biogenesis, there is currently a lack of functional and structural information on the lipoprotein components of the BAM complex. Here, we report the first crystal structure of BamB, the largest and most functionally characterized lipoprotein component of the BAM complex. The crystal structure shows that BamB has an eight-bladed β-propeller structure, with four β-strands making up each blade. Mapping onto the structure the residues previously shown to be important for BamA interaction reveals that these residues, despite being far apart in the amino acid sequence, are localized to form a continuous solvent-exposed surface on one side of the β-propeller. Found on the same side of the β-propeller is a cluster of residues conserved among BamB homologs. Interestingly, our structural comparison study suggests that other proteins with a BamB-like fold often participate in protein or ligand binding, and that the binding interface on these proteins is located on the surface that is topologically equivalent to where the conserved residues and the residues that are important for BamA interaction are found on BamB. Our structural and bioinformatic analyses, together with previous biochemical data, provide clues to where the BamA and possibly a substrate interaction interface may be located on BamB.     

Structural characterization of Escherichia coli BamE, a lipoprotein component of the β-barrel assembly machinery complex

In Escherichia coli, the BAM complex catalyzes the essential process of assembling outer membrane proteins (OMPs). This complex consists of five proteins: one membrane-bound protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their importance in OMP biogenesis, there is currently a lack of functional and structural information on the BAM complex lipoproteins. BamE is the smallest but most conserved lipoprotein in the complex. The structural and dynamic properties of monomeric BamE (residues 21-133) were determined by NMR spectroscopy. The protein folds as two α-helices packed against a three-stranded antiparallel β-sheet. The N-terminal (Ser21-Thr39) and C-terminal (Pro108-Asn113) residues, as well as a β-hairpin loop (Val76-Gln89), are highly flexible on the subnanosecond time scale. BamE expressed and purified from E. coli also exists in a kinetically trapped dimeric state that has dramatically different NMR spectra, and hence structural features, relative to its monomeric form. The functional significance of the BamE dimer remains to be established. Structural comparison to proteins with a similar architecture suggests that BamE may play a role in mediating the association of the BAM complex or with the BAM complex substrates.     

Mechanical unfolding of a β-barrel membrane protein by single-molecule force spectroscopy

正Dear Editor.Transmembrane proteins withβ-barrel topology are mainly found in the outer membranes (OMs) of Gram-negative bacteria,mitochondria and chloroplasts (Wimley,2003).These proteins usually contain even numbers ofβ-strands,ranging from 8-36.To achieve an overall cylindrical topology,the polypeptide chain of aβ-barrel OMP must fold to form a series of anti-parallelβ-strands with eachβ-strand hydrogen-bonding to its neighboring strands (Otzen and Andersen,2013).The folding and insertion of aβ-barrel OMP in vivo requires an evolutionarily conserved multiprotein complex termedβ-barrel assembly machinery(BAM) complex (Noinaj et al.,2015).The structures of the     

The signal distinguishing between targeting of outer membrane β-barrel protein to plastids and mitochondria in plants

The proteome of the outer membrane of mitochondria and chloroplasts consists of membrane proteins anchored by α-helical or β-sheet elements. While proteins with α-helical transmembrane domains are present in all cellular membranes, proteins with β-barrel structure are specific for these two membranes. The organellar β-barrel proteins are encoded in the nuclear genome and thus, have to be targeted to the outer organellar membrane where they are recognized by surface exposed translocation complexes. In the last years, the signals that ensure proper targeting of these proteins have been investigated as essential base for an understanding of the regulation of cellular protein distribution. However, the organellar β-barrel proteins are unique as most of them do not contain a typical targeting information in form of an N-terminal cleavable targeting signal. Recently, it was discovered that targeting and surface recognition of mitochondrial β-barrel proteins in yeast, humans and plants depends on the hydrophobicity of the last β-hairpin of the β-barrel. However, we demonstrate that the hydrophobicity is not sufficient for the discrimination of targeting to chloroplasts or mitochondria. By domain swapping between mitochondrial and chloroplast targeted β-barrel proteins atVDAC1 and psOEP24 we demonstrate that the presence of a hydrophilic amino acid at the C-terminus of the penultimate β-strand is required for mitochondrial targeting. A mutation of the chloroplast β-barrel protein psOEP24 which mimics such profile is efficiently targeted to mitochondria. Thus, we present the properties of the signal for mitochondrial targeting of β-barrel proteins in plants.     

The essential β-barrel assembly machinery complex components BamD and BamA are required for autotransporter biogenesis

Autotransporter biogenesis is dependent upon BamA, a central component of the β-barrel assembly machinery (BAM) complex. In this report, we detail the role of the other BAM components (BamB-E). We identify the importance of BamD in autotransporter biogenesis and show that BamB, BamC, and BamE are not required.