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.     

Neisserial Omp85 protein is selectively recognized and assembled into functional complexes in the outer membrane of human mitochondria

As a consequence of their bacterial origin, mitochondria contain β-barrel proteins in their outer membrane (OMM). These proteins require the translocase of the outer membrane (TOM) complex and the conserved sorting and assembly machinery (SAM) complex for transport and integration into the OMM. The SAM complex and the β-barrel assembly machinery (BAM) required for biogenesis of β-barrel proteins in bacteria are evolutionarily related. Despite this homology, we show that bacterial β-barrel proteins are not universally recognized and integrated into the OMM of human mitochondria. Selectivity exists both at the level of the TOM and the SAM complex. Of all of the proteins we tested, human mitochondria imported only β-barrel proteins originating from Neisseria sp., and only Omp85, the central component of the neisserial BAM complex, integrated into the OMM. PorB proteins from different Neisseria, although imported by the TOM, were not recognized by the SAM complex and formed membrane complexes only when functional Omp85 was present at the same time in mitochondria. Omp85 alone was capable of integrating other bacterial β-barrel proteins in human mitochondria, but could not substitute for the function of its mitochondrial homolog Sam50. Thus, signals and machineries for transport and assembly of β-barrel proteins in bacteria and human mitochondria differ enough to allow only a certain type of β-barrel proteins to be targeted and integrated in mitochondrial membranes in human cells.     

Escherichia coli outer membrane protein A adheres to human brain microvascular endothelial cells

Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis. The outer membrane protein A (OmpA) assembles a beta-barrel structure having four surface-exposed loops in E. coli outer membrane. OmpA of meningitis-causing E. coli K1 is shown to contribute to invasion of the human brain microvascular endothelial cells (HBMEC), the main cellular component of the blood-brain barrier (BBB). However, the direct evidence of OmpA protein interacting with HBMEC is not clear. In this study, we showed that OmpA protein, solubilized from the outer membrane of E. coli, adhered to HBMEC surface. To verify OmpA interaction with the HBMEC, we purified N-terminal membrane-anchoring beta-barrel domain of OmpA and all surface-exposed loops deleted OmpA proteins, and showed that the surface-exposed loops of OmpA were responsible for adherence to HBMEC. These findings indicate that the OmpA is the adhesion molecule with HBMEC and the surface-exposed loops of OmpA are the determinant of this interaction.     

Expression of outer membrane proteins by Salmonella enteritidis relating to pH

Abstract The pH of the environment influenced the expression of outer membrane protein by S. enteritidis PT4 growing in broth. Growth in broth at pH 5 to 7 resulted in variation in expression of outer membrane proteins of 18 to 22 kDa. Bacteria became acid-fixed and non-viable following prolonged incubation in broth with a pH below 5, and expression of flagella was repressed.     

Determining OMP topology by computation, surface plasmon resonance and cysteine labelling: the test case of OMPG

Bacterial outer-membrane proteins (OMP) are important in pathogenicity and the recently solved structure of OmpG provides an excellent test case for topological predictions since it is monomeric. Here we compare the results of applying several computerised structure prediction algorithms to the sequence of OmpG. Furthermore, we probe the OmpG topology by both an established chemical labelling approach and a new method which combines epitope insertion and surface plasmon resonance. The computational approaches are broadly accurate but the exact choice of the number of beta strands remains difficult. The algorithms also tend to predict the entire beta strand rather than just the transmembrane region. Epitope insertion clearly pinpoints exposed loops but its utility in defining buried or periplasmic sites is less clear cut. Cysteine-mutant labelling is largely confined to exposed residues but one periplasmic cysteine may be labelled by reagents entering via the OmpG pore.     

Chloroplast β-Barrel Proteins Are Assembled into the Mitochondrial Outer Membrane in a Process That Depends on the TOM and TOB Complexes

Membrane-embedded β-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. In eukaryotic cells, precursors of these proteins are synthesized in the cytosol and have to be sorted to their corresponding organelle. Currently, the signal that ensures their specific targeting to either mitochondria or chloroplasts is ill-defined. To address this issue, we studied targeting of the chloroplast β-barrel proteins Oep37 and Oep24. We found that both proteins can be integrated in vitro into isolated plant mitochondria. Furthermore, upon their expression in yeast cells Oep37 and Oep24 were exclusively located in the mitochondrial OM. Oep37 partially complemented the growth phenotype of yeast cells lacking Porin, the general metabolite transporter of this membrane. Similarly to mitochondrial β-barrel proteins, Oep37 and Oep24 expressed in yeast cells were assembled into the mitochondrial OM in a pathway dependent on the TOM and TOB complexes. Taken together, this study demonstrates that the central mitochondrial components that mediate the import of yeast β-barrel proteins can deal with precursors of chloroplast β-barrel proteins. This implies that the mitochondrial import machinery does not recognize signals that are unique to mitochondrial β-barrel proteins. Our results further suggest that dedicated targeting factors had to evolve in plant cells to prevent mis-sorting of chloroplast β-barrel proteins to mitochondria.     

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

The proteolytic fragments of OprFs of Pseudomonas aeruginosa and Pseudomonas fluorescens were identified, respectively, as the first 175 and 177 amino acids from the N-terminal domain. They induced ion channels after reincorporation into planar lipid bilayers (85 and 75 pS, respectively, in 1 M NaCl). A similar conductance value (72 pS) was found for the eight beta-strand OmpA N-terminal domain (OmpA171) of Escherichia coli. We conclude that the N-terminal domain of OprFs is sufficient to induce ion channels and the comparison with OmpA171, provides strong evidence of the existence of an eight-stranded beta-barrel in the N-terminal domain of OprFs.     

Cloning and Characterization of a 35-kDa Mouse Mitochondrial Outer Membrane Protein MOM35 with High Homology to Tom40

We have cloned a 35-kDa protein from a mouse cDNA library with a 25% overall amino acididentity to yTom40 and 27% identity to nTom40. This homolog to Tom40 was named MOM35.It contains two possible start codons 36 amino acids apart from each other. Both the long andthe short version of MOM35 can be imported in vitro into mouse mitochondria. The identifiedprotein is imported into the outer mitochondrial membrane and comprises a trypsin-resistancepattern similar to that of nTom40. Tom40 of N. crassa, S. cerevisiae, and the protein identifiedherein contains a highly conserved region with possible physiological importance. Subsequentinvestigation has revealed that this region interacts specifically in vitro with preproteinsproposed to be imported by a Tom40-dependent pathway.     

Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly

Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.     

Evidence Supporting the 19 β-Strand Model for Tom40 from Cysteine Scanning and Protease Site Accessibility Studies

Most proteins found in mitochondria are translated in the cytosol and enter the organelle via the TOM complex (translocase of the outer mitochondrial membrane). Tom40 is the pore forming component of the complex. Although the three-dimensional structure of Tom40 has not been determined, the structure of porin, a related protein, has been shown to be a β-barrel containing 19 membrane spanning β-strands and an N-terminal α-helical region. The evolutionary relationship between the two proteins has allowed modeling of Tom40 into a similar structure by several laboratories. However, it has been suggested that the 19-strand porin structure does not represent the native form of the protein. If true, modeling of Tom40 based on the porin structure would also be invalid. We have used substituted cysteine accessibility mapping to identify several potential β-strands in the Tom40 protein in isolated mitochondria. These data, together with protease accessibility studies, support the 19 β-strand model for Tom40 with the C-terminal end of the protein localized to the intermembrane space.     

Outer membrane proteins: comparing X-ray and NMR structures by MD simulations in lipid bilayers

The structures of three bacterial outer membrane proteins (OmpA, OmpX and PagP) have been determined by both X-ray diffraction and NMR. We have used multiple (7 × 15 ns) MD simulations to compare the conformational dynamics resulting from the X-ray versus the NMR structures, each protein being simulated in a lipid (DMPC) bilayer. Conformational drift was assessed via calculation of the root mean square deviation as a function of time. On this basis the ‘quality’ of the starting structure seems mainly to influence the simulation stability of the transmembrane β-barrel domain. Root mean square fluctuations were used to compare simulation mobility as a function of residue number. The resultant residue mobility profiles were qualitatively similar for the corresponding X-ray and NMR structure-based simulations. However, all three proteins were generally more mobile in the NMR-based than in the X-ray simulations. Principal components analysis was used to identify the dominant motions within each simulation. The first two eigenvectors (which account for >50% of the protein motion) reveal that such motions are concentrated in the extracellular loops and, in the case of PagP, in the N-terminal α-helix. Residue profiles of the magnitude of motions corresponding to the first two eigenvectors are similar for the corresponding X-ray and NMR simulations, but the directions of these motions correlate poorly reflecting incomplete sampling on a ∼10 ns timescale.     

Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae

Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae. A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described. The method begins with intact H. influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated. P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation. The procedure yields highly purified P6. Contamination with lipooligosaccharide is less than 0.025 endotoxin U per microgr P6. The yield of P6 is approximately 2 mg of P6 per l H. influenzae culture. The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies. The purified P6 is immunogenic in animals. A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6.     

Bacterial Origin of a Mitochondrial Outer Membrane Protein Translocase: NEW PERSPECTIVES FROM COMPARATIVE SINGLE CHANNEL ELECTROPHYSIOLOGY

Mitochondria are of bacterial ancestry and have to import most of their proteins from the cytosol. This process is mediated by Tom40, an essential protein that forms the protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic translocase of the outer mitochondrial membrane (ATOM), a protein that shows similarities to both eukaryotic Tom40 and bacterial protein translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other protein translocases reveals that ATOM closely resembles bacterial-type protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes.     

Proteome analysis of mitochondrial outer membrane from Neurospora crassa

The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of LC-MS/MS of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS PMF. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.     

Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.     

Fluorescence-mediated analysis of mitochondrial preprotein import in vitro

Mitochondrial biogenesis is a crucial element of the functional maintenance of a eukaryotic cell. The organelle must import the majority of its proteins from the cytosol where they are synthesized as precursors. In vitro import assays have been developed in which isolated mitochondria are incubated with precursor proteins, that are generated either by in vitro translation systems or by expression and purification as recombinant proteins. The detection of imported proteins is performed by autoradiography or by Western blot. We have now established a novel detection system for imported precursor proteins that is based on fluorescent labeling. We constructed a mitochondrial preprotein containing a C-terminal SNAP-tag that can label itself with a single fluorescein molecule in an enzymatic reaction. The fluorescent preproteins were efficiently imported into isolated mitochondria and showed kinetic behavior similar to that of standard preproteins. The fluorescence detection was sensitive and significantly faster than other comparable procedures. We also showed that precursor proteins containing a SNAP-tag domain could be successfully labeled in a postimport reaction in intact mitochondria. In summary, the use of a reporter domain modified with a fluorescent dye provides a novel, sensitive, and fast detection method to analyze the properties of the mitochondrial import reaction in vitro.     

The plant mitochondrial protein import apparatus — The differences make it interesting

Background

Mitochondria play essential roles in the life and death of almost all eukaryotic cells, ranging from single-celled to multi-cellular organisms that display tissue and developmental differentiation. As mitochondria only arose once in evolution, much can be learned from studying single celled model systems such as yeast and applying this knowledge to other organisms. However, two billion years of evolution have also resulted in substantial divergence in mitochondrial function between eukaryotic organisms.

Scope of Review

Here we review our current understanding of the mechanisms of mitochondrial protein import between plants and yeast (Saccharomyces cerevisiae) and identify a high level of conservation for the essential subunits of plant mitochondrial import apparatus. Furthermore, we investigate examples whereby divergence and acquisition of functions have arisen and highlight the emerging examples of interactions between the import apparatus and components of the respiratory chain.

Major conclusions

After more than three decades of research into the components and mechanisms of mitochondrial protein import of plants and yeast, the differences between these systems are examined. Specifically, expansions of the small gene families that encode the mitochondrial protein import apparatus in plants are detailed, and their essential role in seed viability is revealed.

General significance

These findings point to the essential role of the inner mitochondrial protein translocases in Arabidopsis, establishing their necessity for seed viability and the crucial role of mitochondrial biogenesis during germination. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.