Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.     

The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (K_d) determined were 1.08 ± 0.49 and 0.0086 ± 0.0006 μM, respectively, consistent with previously reported values. Furthermore, binding affinities (K_i) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained K_i values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.     

Structure of the glycoprotein Ib.IX complex from platelet membranes

The glycoprotein Ib.IX complex is a major component of the platelet membrane. It mediates the adhesion of platelets to exposed subendothelium and provides an attachment site for the membrane skeleton on the plasma membrane. The present study was designed to characterize the structure of the glycoprotein Ib.IX complex. Electron microscopy of purified glycoprotein Ib.IX complex in detergent showed that each complex existed as a flexible rod with a globular domain on either end. The overall length of the complex was approximately 59.5 nm. The smaller globular domain had a diameter of approximately 8.9 nm; the larger, a diameter of approximately 15.9 nm. In the absence of detergent, the glycoprotein Ib.IX complexes tended to self-associate through the larger globular domain, suggesting that this domain contained the hydrophobic region that inserts into the membrane. Proteases known to cleave glycoprotein Ib alpha close to its membrane-insertion site released the larger globular domain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that this domain was composed of glycoprotein Ib beta, glycoprotein IX, and a Mr = 25,000 fragment of glycoprotein Ib alpha. Proteolysis at the external end of glycoprotein Ib alpha reduced the size of the smaller globular domain. This study shows that the glycoprotein Ib.IX complex has an elongated shape, with a globular domain on the end that inserts into the membrane and a smaller globular domain on the end of glycoprotein Ib alpha that is oriented external to the plasma membrane.     

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence,gliding motility and the degradation of complex biopolymers

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres‐Chlorobi‐Bacteroidetes superphylum. Proteins secreted by the T9SS have an N‐terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C‐terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C‐terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components.     

Electron-microscopic localization of type II,IX, and V collagen in the organ of Corti of the gerbil

Summary The presence of types II, IX and V collagen was probed in the organ of Corti of the adult gerbil cochlea by use of immunocytochemistry at the light- and electron-microscopic levels. Type II collagen is found in the connective tissues of the osseous spiral lamina and spiral limbus. In the region of the sensory hair cells it is present in the tectorial membrane and antibodies bind to the thick unbranched radial fibers. Type IX collagen co-localizes with type II collagen in the tectorial membrane, where antibodies bind to the thick unbranched radial fibers. Type V collagen is present in the connective tissue of the spiral limbus, the osseous spiral lamina, the eighth nerve, and the tectorial membrane. In the tectorial membrane, the staining with antibodies to type V collagen is more diffuse than that seen for types II and IX collagen and antibodies to type V bind to the thin, highly branched fibers in which the thick fibers are embedded. The results indicate that collagens characteristic of cartilage are localized in the organ of Corti. Within the tectorial membrane, types II and IX collagen form heterotypic thick fibers embedded in a reticular network of type V collagen fibers. These collagens form a highly structured matrix which contributes to the rigidity of the tectorial membrane and allow it to withstand the physical stresses associated with transmission of the stimuli necessary for sensory transduction.     

Detection of protoporphyrin IX in envelope membranes of pea chloroplasts

Envelope membranes were prepared from mature pea chloroplasts. The tetrapyrrole contents of envelope membranes were analysed. The envelope membranes of pea chloroplasts contained substantial amounts of protoporphyrin IX and trace amounts of Mg-protoporphyrin IX and its monoester in addition to protochlorophyllide. The protoporphyrin IX content of envelope membranes was 89.25 pmol (mg protein)(-1). Its content in pea envelope membrane was higher than that of protochlorophyllide. The proportion of monovinyl and divinyl forms of protochlorophyllide present in pea chloroplast envelope membrane was 3:7. The significance of the presence of protoporphyrin IX in the envelope membrane is discussed in relation to plastidic Chl biosynthesis.     

Transmembrane segment (TMS) VIII of the Na(+)/Citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane.

The amino acid sequence of the sodium ion-dependent citrate transporter CitS of K. pneumoniae contains 12 hydrophobic stretches that could form membrane-spanning segments. A previous analysis of the membrane topology in Escherichia coli using the PhoA gene fusion technique indicated that only nine of these hydrophobic segments span the membrane, while three segments, Vb, VIII and IX, were predicted to have a periplasmic location (Van Geest, M., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589). A topology study of C-terminally truncated CitS molecules in dog pancreas microsomes revealed that the protein traverses the endoplasmic reticulum membrane 11 times. In agreement with the PhoA fusion data, segment Vb was predicted to have a periplasmic location, but, in contrast, segments VIII and IX were found to be membrane-spanning (Van Geest, M., Nilsson, I., von Heijne, G., and Lolkema, J. S. (1999) J. Biol. Chem. 274, 2816-2823). In the present study, using site-directed Cys labeling, the topology of segments VIII and IX in the full-length CitS protein was determined in the E. coli membrane. Engineered cysteine residues in the loop between the two segments were accessible to a membrane-impermeable thiol reagent exclusively from the cytoplasmic side of the membrane, demonstrating that transmembrane segments (TMSs) VIII and IX are both membrane-spanning. It follows that the folding of CitS in the E. coli and endoplasmic reticulum membrane is the same. Cysteine accessibility studies of CitS-PhoA fusion molecules demonstrated that in the E. coli membrane segment VIII is exported to the periplasm in the absence of the C-terminal CitS sequences, thus explaining why the PhoA fusions do not correctly predict the topology. An engineered cysteine residue downstream of TMS VIII moved from a periplasmic to a cytoplasmic location when the fusion protein containing TMSs I-VIII was extended with segment IX. Thus, downstream segment IX is both essential and sufficient for the insertion of segment VIII of CitS in the E. coli membrane.     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

Chloroplast Biogenesis 65 : Enzymic Conversion of Protoporphyrin IX to Mg-Protoporphyrin IX in a Subplastidic Membrane Fraction of Cucumber Etiochloroplasts

The preparation from Percoll-purified cucumber (Cucumis sativus)etiochloroplasts of a subplastidic membrane fraction that is capable of high rates of Mg insertion into protoporphyrin IX is described. The plastid stroma was inactive when used either alone or in combination with the membrane fraction. Successful preparation of the subplastidic membrane fraction required that Mg-protoporphyrin chelatase was first stabilized by its substrate. This was achieved by lysing Percoll-purified plastids in a fortified hypotonic medium containing protoporphyrin IX prior to ultracentrifugation and separation of the stroma from the plastid membranes. Protoporphyrin IX became membrane bound. Other additives needed for enzyme activity fell into two groups: (a) those needed for enzyme stabilization during membrane preparation and (b) those involved in the primary mechanism of Mg insertion into protoporphyrin IX. Ethylenediaminetetraacetate belonged to the first group, magnesium belonged to the second group, and ATP belonged to both groups.     

Evidence for activation of tissue factor by an allosteric disulfide bond

Tissue Factor (TF) is the mammalian plasma membrane cofactor responsible for initiation of blood coagulation. Binding of blood coagulation factor VIIa to TF activates the serine proteinase zymogens factors IX and X by limited proteolysis leading to the formation of a thrombin and fibrin meshwork that stabilizes the thrombus. TF on the plasma membrane of cells resides mostly in a cryptic configuration, which rapidly transforms into an active configuration in response to certain stimuli. The extracellular part of TF consists of two fibronectin type III domains. The disulfide bond in the membrane proximal domain (Cys186-Cys209) is atypical for domains of this type in that it links adjacent strands in the same beta sheet, what we have called an allosteric bond. Ablation of the allosteric disulfide by mutating both cysteine residues severely impairs procoagulant activity. The thiol-alkylating agents N-ethylmaleimide and methyl methanethiolsulfonate block TF activation by ionomycin, while the thiol-oxidizing agent HgCl2 and dithiol cross-linkers promote activation. TF activation could not be explained by exposure of phosphatidylserine on the outer leaflet of the plasma membrane. Cryptic TF contained unpaired cysteine thiols that were depleted upon activation, and de-encryption was associated with a change in the conformation of the membrane-proximal domain. These findings imply that the Cys186-Cys209 disulfide bond is reduced in the cryptic form of TF and that activation involves formation of the disulfide. It is likely that formation of this disulfide bond changes the conformation of the domain that facilitates productive binding of factors IX and X.     

Crystal structure of protoporphyrinogen oxidase from Myxococcus xanthus and its complex with the inhibitor acifluorfen

Protoporphyrinogen IX oxidase, a monotopic membrane protein, which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the heme/chlorophyll biosynthetic pathway, is distributed widely throughout nature. Here we present the structure of protoporphyrinogen IX oxidase from Myxococcus xanthus, an enzyme with similar catalytic properties to human protoporphyrinogen IX oxidase that also binds the common plant herbicide, acifluorfen. In the native structure, the planar porphyrinogen substrate is mimicked by a Tween 20 molecule, tracing three sides of the macrocycle. In contrast, acifluorfen does not mimic the planarity of the substrate but is accommodated by the shape of the binding pocket and held in place by electrostatic and aromatic interactions. A hydrophobic patch surrounded by positively charged residues suggests the position of the membrane anchor, differing from the one proposed for the tobacco mitochondrial protoporphyrinogen oxidase. Interestingly, there is a discrepancy between the dimerization state of the protein in solution and in the crystal. Conserved structural features are discussed in relation to a number of South African variegate porphyria-causing mutations in the human enzyme.     

Nature of the regions involved in the insertion of newly synthesized protein into the outer membrane of Escherichia coli.

Outer membrane proteins are synthesized by cytoplasmic membrane-bound polysomes, and inserted at insertion sites which cover about 10% of the total outer membrane when cells grow with a generation time of 1 h. A membrane fraction enriched in outer membrane insertion regions was isolated and partly characterized. The rat at which newly inserted proteins are transferred from such insertion regions into the rest of the outer membrane was found to be very fast; the new protein content of insertion regions and that of the remaining outer membrane equilibrate completely within about 20 s at 25 degrees C. Given the rather rigid structure of the outer membrane and the multiple interactions between outer membrane components and the murein layer, lateral diffusion of newly inserted proteins from insertion sites to the remaining outer membrane is not likely to explain this rapid equilibration. Instead, the data support a model in which insertion regions move along the cell surface, leaving behind stationary, newly inserted outer membrane proteins.     

Biosynthesis of type IX collagen during chick limb development

We analyzed the collagens synthesized by developing chick limbs (stages 22 to 34). Type IX collagen synthesis started at stage 26, concurrently with the chondrogenic differentiation of limb mesenchyme, and gradually increased during subsequent stages. By stage 34, the central cartilaginous region of the limbs substantially synthesized type IX collagen, in addition to cartilage-specific type II collagen, while the outer non-cartilaginous region of the limbs synthesized predominantly type I collagen. The present study indicates that type IX collagen is cartilage-specific and can be used as a marker for the chondrogenic phenotype.     

Nature of the regions involved in the insertion of newly synthesized protein into the outer membrane of Escherichia coli

Outer membrane proteins are synthesized by cytoplasmic membrane-bound polysomes, and inserted at insertion sites which cover about 10% of the total outer membrane when cells grow with a generation time of 1 h. A membrane fraction enriched in outer membrane insertion regions was isolated and partly characterized. The rate at which newly inserted proteins are transferred from such insertion regions into the rest of the outer membrane was found to be very fast; the new protein content of insertion regions and that of the remaining outer membrane equilibrate completely within about 20 s at 25°C.Given the rather rigid structure of the outer membrane and the multiple interactions between outer membane components and the murein layer, lateral diffusion of newly inserted proteins from insertion sites to the remaining outer membrane is not likely to explain this rapid equilibration. Instead, the data support a model in which mobile insertion regions move along the cell surface, leaving behind stationary, newly inserted outer membrane proteins.     

Characterization of the LolA-LolB system as the general lipoprotein localization mechanism of Escherichia coli.

The major outer membrane lipoprotein (Lpp) of Escherichia coli requires LolA for its release from the cytoplasmic membrane, and LolB for its localization to the outer membrane. We examined the significance of the LolA-LolB system as to the outer membrane localization of other lipoproteins. All lipoproteins possessing an outer membrane-directed signal at the N-terminal second position were efficiently released from the inner membrane in the presence of LolA. Some lipoproteins were released in the absence of externally added LolA, albeit at a slower rate and to a lesser extent. This LolA-independent release was also strictly dependent on the outer membrane sorting signal. A lipoprotein-LolA complex was formed when the release took place in the presence of LolA, whereas lipoproteins released in the absence of LolA existed as heterogeneous complexes, suggesting that the release and the formation of a complex with LolA are distinct events. The release of LolB, an outer membrane lipoprotein functioning as the receptor for a lipoprotein-LolA complex, occurred with a trace amount of LolA, and therefore was extremely efficient. The LolA-dependent release of lipoproteins was found to be crucial for the specific incorporation of lipoproteins into the outer membrane, whereas lipoproteins released in the absence of LolA were nonspecifically and inefficiently incorporated into the membrane. The outer membrane incorporation of lipoproteins including LolB per se was dependent on LolB in the outer membrane. From these results, we conclude that lipoproteins in E. coli generally utilize the LolA-LolB system for efficient release from the inner membrane and specific localization to the outer membrane.     

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide

We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig. We conclude that selective NRB transport into rat mitochondria occurs via TSPO in the OMM, which allows its translocation to PT-regulating sites in the inner membrane. Thus, species-specificity of NRB toward the rat PT depends on subtle differences in the structure of TSPO or of TSPO-associated proteins affecting its substrate specificity.     

Localization of proteolytic activity in the outer membrane of Escherichia coli.

An enzyme in the cytoplasmic membrane, nitrate reductase, can be solubilized by heating membranes to 60 degrees C for 10 min at alkaline pH. A protease in the cell envelope has been shown to be responsible for this solubilization. The localization of this protease in the outer membrane was demonstrated by separating the outer membrane from the cytoplasmic membrane, adding back various forms of outer membrane protein to the cytoplasmic membrane, and following the increase in nitrate reductase solubilization with increasing amounts of outer membrane proteins. This solubilization is accompanied by the cleavage of one of the subunits of nitrate reductase and is inhibited by the protease inhibitor p-aminobenzamidine. Analysis of membrane proteins synthesized by cells grown in the presence of various amounts of p-aminobenzamidine revealed that p-aminobenzamidine affects the synthesis of the major outer membrane proteins but has little effect on the synthesis of cytoplasmic membrane proteins. When outer membrane is reacted with the protease inhibitor [3H]diisopropylfluorophosphate, a single protein in the outer membrane is labeled. Since the interaction with diisopropylfluorophosphate is inhibited by p-aminobenzamidine, it is suggested that this single outer membrane protein is responsible for the in vitro solubilization of nitrate reductase and the in vivo processing of the major outer membrane proteins.     

High‐resolution architecture of the outer membrane of the Gram‐negative bacteria Roseobacter denitrificans

The outer membrane of Gram‐negative bacteria protects the cell against bactericidal substances. Passage of nutrients and waste is assured by outer membrane porins, beta‐barrel transmembrane channels. While atomic structures of several porins have been solved, so far little is known on the supramolecular structure of the outer membrane. Here we present the first high‐resolution view of a bacterial outer membrane gently purified maintaining remnants of peptidoglycan on the perisplasmic surface. Atomic force microscope images of outer membrane fragments of the size of ～50% of the bacterial envelope revealed that outer membrane porins are by far more densely packed than previously assumed. Indeed the outer membrane is a molecular sieve rather than a membrane. Porins cover ～70% of the membrane surface and form locally regular lattices. The potential role of exposed aromatic residues in the formation of the supramolecular assembly is discussed. Finally, we present first structural data of the outer membrane porin from the marine Gram‐negative bacteria Roseobacter denitrificans, and we perform a sequence alignment with porins of known structure.     

Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae

The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.     

TonB interacts with nonreceptor proteins in the outer membrane of Escherichia coli

The Escherichia coli TonB protein serves to couple the cytoplasmic membrane proton motive force to active transport of iron-siderophore complexes and vitamin B(12) across the outer membrane. Consistent with this role, TonB has been demonstrated to participate in strong interactions with both the cytoplasmic and outer membranes. The cytoplasmic membrane determinants for that interaction have been previously characterized in some detail. Here we begin to examine the nature of TonB interactions with the outer membrane. Although the presence of the siderophore enterochelin (also known as enterobactin) greatly enhanced detectable cross-linking between TonB and the outer membrane receptor, FepA, the absence of enterochelin did not prevent the localization of TonB to the outer membrane. Furthermore, the absence of FepA or indeed of all the iron-responsive outer membrane receptors did not alter this association of TonB with the outer membrane. This suggested that TonB interactions with the outer membrane were not limited to the TonB-dependent outer membrane receptors. Hydrolysis of the murein layer with lysozyme did not alter the distribution of TonB, suggesting that peptidoglycan was not responsible for the outer membrane association of TonB. Conversely, the interaction of TonB with the outer membrane was disrupted by the addition of 4 M NaCl, suggesting that these interactions were proteinaceous. Subsequently, two additional contacts of TonB with the outer membrane proteins Lpp and, putatively, OmpA were identified by in vivo cross-linking. These contacts corresponded to the 43-kDa and part of the 77-kDa TonB-specific complexes described previously. Surprisingly, mutations in these proteins individually did not appear to affect TonB phenotypes. These results suggest that there may be multiple redundant sites where TonB can interact with the outer membrane prior to transducing energy to the outer membrane receptors.