ATP-dependent ferric hydroxamate transport system in Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a transmembrane/cytoplasmic region of the integral membrane protein FhuB, as revealed by competitive peptide mapping

The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes. Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cytoplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane. Interaction of FhuD with FhuB was studied in vitro with biotinylated synthetic 10 residue and 20–24 residue peptides of FhuB by determining the activity of β-galactosidase linked to the peptides via streptavidin. Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and part of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD. Decapeptides were transferred into the periplasm of cells through a FhuA deletion derivative that forms permanently open channels three times as large as the porins in the outer membrane. FhuB peptides that bound to FhuD inhibited ferrichrome transport, while peptides that did not bind to FhuD did not affect transport. These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytoplasmic segment 7 of FhuB. The transmembrane region may be part of a pore through which a portion of FhuD inserts into the cytoplasmic membrane during transport. The cytoplasmic segment 7 of FhuB contains the conserved amino acid sequence EAA…G (in FhuB DTA…G) found in ABC transporters, which is predicted to interact with the cytoplasmic FhuC ATPase. Triggering of ATP hydrolysis by substrate-loaded FhuD may occur by physical interaction between FhuD and FhuC, which bind close to each other on loop 7. Although FhuB consists of two homologous halves, FhuB(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome transport are asymmetrically arranged.     

Iron(III) hydroxamate transport across the cytoplasmic membrane ofEscherichia coli

Summary Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm ofEscherichia coli is mediated by the FhuC, FhuD and FhuB proteins and displays characteristics typical of a periplasmic-binding-protein-dependent transport mechanism. In contrast to the highly specific receptor proteins in the outer membrane, at least six different siderophores of the hydroxamate type and the antibiotic albomycin are accepted as substrates. AfhuB mutant (deficient in transport of substrates across the inner membrane) which overproduced the periplasmic FhuD 30-kDa protein, bound [⁵⁵Fe] iron(III) ferrichrome. Resistance of FhuD to proteinase K in the presence of ferrichrome, aerobactin, and coprogen indicated binding of these substrates to FhuD. FhuD displays significant similarity to the periplasmic FecB, FepB, and BtuE proteins. The extremely hydrophobic FhuB 70-kDa protein is located in the cytoplasmic membrane and consists of two apparently duplicated halves. The N-and C-terminal halves [FhuB(N) and FhuB(C)] were expressed separately infhuB mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamate as a sole iron source, indicating that both halves of FhuB were essential for substrate translocation and that they combined to form an active permease. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be transport-active, indicating that the extra portion did not disturb proper insertion of the active FhuB segments into the cytoplasmic membrane. A region of considerable similarity, present twice in FhuB, was identified near the C-terminus of 20 analyzed hydrophobic proteins of periplasmic-binding-protein-dependent systems. The FhuC 30 kDa protein, most likely involved in ATP binding, contains two domains representing consensus sequences among all peripheral cytoplasmic membrane proteins of these systems. Amino acid replacements in domain I (LysGlu and Gln) and domain II (AspAsn and Glu) resulted in a transport-deficient phenotype.     

Point mutations in two conserved glycine residues within the integral membrane protein FhuB affect iron(II) hydroxamate transport

Summary A region of substantial homology, comprising 32 amino acids around a highly conserved glycine residue, is located near the C-terminal ends of the hydrophobic Fhu, Fec, Fep, Fat, and Btu transport proteins involved in the uptake of ferrisiderophores and vitamin B₁₂ into Escherichia coli and Vibrio anguillarum. Furthermore, a region similar in location and sequence containing an invariant glycine at an equivalent position was identified in the hydrophobic component of all other periplasmic binding protein-dependent (PBT) systems. In the FhuB protein, which is twice the size of the other PBT-related inner membrane proteins and which displays an internal homology, two conserved glycine residues are present. Alteration of Gly at positions 226 and 559 to Ala, Val, or Glu reduced iron(III) hydroxamate uptake, suggesting that this homologous region may play a general role in the mechanism of PBT-dependent transport.     

Iron(III) hydroxamate transport of Escherichia coli: restoration of iron supply by coexpression of the N- and C-terminal halves of the cytoplasmic membrane protein FhuB cloned on separate plasmids

Summary Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm of Escherichia coli cells is mediated by the FhuC, FhuD and FhuB proteins. We studied the extremely hydrophobic FhuB protein (70 kDa) which is located in the cytoplasmic membrane. The N- and C-terminal halves of the protein [FhuB(N) and FhuB(C)] show homology to each other and to the equivalent polypeptides involved in uptake of ferric dicitrate and of vitamin B₂. Various plasmids carrying only one-half of the fhuB gene were expressed in fhuB ^– mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamates as sole iron source; no activity was obtained with either half of FhuB alone. These results indicate that both halves of FhuB are essential for substrate translocation and that they combine to form an active permease when expressed separately. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be partially active in transport, indicating that the extra portion did not perurb proper insertion of the active FhuB segments into the cytoplasmic membrane.     

Iron(III) hydroxamate transport in Escherichia coli K-12: FhuB-mediated membrane association of the FhuC protein and negative complementation of fhuC mutants.

Iron(III) hydroxamate transport across the cytoplasmic membrane is catalyzed by the very hydrophobic FhuB protein and the membrane-associated FhuC protein, which contains typical ATP-binding domains. Interaction between the two proteins was demonstrated by immunoelectron microscopy with anti-FhuC antibodies, which showed FhuB-mediated association of FhuC with the cytoplasmic membrane. In addition, inactive FhuC derivatives carrying single amino acid replacements in the ATP-binding domains suppressed wild-type FhuC transport activity, which arose either from displacement of active FhuC from FhuB by the mutated FhuC derivatives or from the formation of mixed inactive FhuC multimers between wild-type and mutated FhuC proteins. Inactive FhuC derivatives containing internal deletions and insertions showed no phenotypic suppression, indicating conformational alterations that rendered the FhuC derivatives unable to displace wild-type FhuC. It is concluded that the physical interaction between FhuC and FhuB implies a coordinate activity of both proteins in the transport of iron(III) hydroxamates through the cytoplasmic membrane.     

Iron-hydroxamate transport into Escherichia coli K12: localization of FhuD in the periplasm and of FhuB in the cytoplasmic membrane

Summary ThefhuB, fhuC andfhuD genes encode proteins which catalyze transport of iron(III)-hydroxamate compounds from the periplasm into the cytoplasm ofEscherichia coli. ThefhuB, C, D genes were cloned downstream of a strong phage T7 promoter and transcribed by T7 RNA polymerase. The overexpressed FhuD protein appeared in two forms of 31 and 28 kDa and was released upon conversion of vegetative cells into spheroplasts, suggesting synthesis of FhuD as a precursor and export into the periplasm. The very hydrophobic FhuB protein was found in the cytoplasmic membrane. These properties, together with the previously found homologies in the FhuC protein to ATP-binding proteins, display the characteristics of a periplasmic binding protein dependent transport system across the cytoplasmic membrane. The molecular weight of FhuB and the sequence offhuC, as previously published by us, was confirmed. FhuB exhibited double the size of most hydrophobic proteins of such systems and showed homology between the amino- and carboxy-terminal halves of the protein, indicating duplication of an original gene and subsequent fusion of the two DNA fragments.     

Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum.

The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.     

Differential Labeling of Components in Human Erythrocyte Membranes Associated with the Transport of Glucose

The irreversible inhibition of glucose transport by 1-fluoro-2,4-dinitrobenzene (FDNB) has been used to identify membrane proteins possibly associated with glucose transport in human crythrocytes. D-Glucose was shown to enhance significantly the rate of FDNB inhibition of transport when present during the reaction, whereas cytochalasin B (CB) and D-maltose retarded this FDNB inhibition of transport. This modulation of the inhibition reaction formed the basis for a double isotopic differential labeling technique using [¹⁴C]- and [³H]FDNB followed by SDS-polyacrylamide gel electrophoresis to distinguish transport-associated polypeptides from bulk membrane dinitrophenylated proteins.

Reactions in the presence of CB or maltose revealed the presence of a differentially labeled polypeptide(s), with a molecular weight of approximately 60,000-65,000 daltons. This effect could in part be reversed in the presence of D-glucose but not L-glucose. Reactions in the presence of D-glucose resulted in two regions of differential labeling. One region was around 200,000 daltons and the other corresponded to a 90,000-dalton band.

Extraction of membrane proteins with p-chloromercuribenzene sulfonate resulted in no loss of the 60,000-dalton peak, indicating that this labeled polypeptide(s) was firmly anchored in the hydrophobic core of the membrane.

These results indicate that as many as three membrane polypeptides are differentially labeled by FDNB under conditions strongly associated with the inhibition of the glucose transport system and may be involved in the regulation of glucose transport.     

Identification of identical binding polypeptides for cephalosporins and dipeptides in intestinal brush-border membrane vesicles by photoaffinity labeling

The uptake of a photolabile derivative of the orally effective cephalosporin cephalexin, N-(4-azidobenzoyl)cephalexin, was investigated in brush-border membrane vesicles. The compound was taken up into the intravesicular space and inhibited the active uptake of cephalexin in a concentration-dependent manner. Therefore, this probe interacts with the transport system shared by alpha-aminocephalosporins and dipeptides. Photoaffinity labeling of brush-border membrane vesicles from rat small intestine with N-(4-azido[3,5-3H]benzoyl) derivatives of the cephalosporin cephalexin and the dipeptide glycyl-L-proline resulted in the covalent incorporation of radioactivity into membrane polypeptides with apparent molecular weights of 127,000, 100,000, 94,000 and 86,000, the polypeptide of molecular weight 127,000 being predominantly labeled. The specificity of labeling was demonstrated by a decrease in the labeling of the polypeptide of apparent molecular weight 127,000 in the presence of beta-lactam antibiotics and dipeptides, whereas glucose, taurocholate or amino acids had no effect on the labeling pattern. These data demonstrate an interaction of cephalosporins and dipeptides with a common membrane protein of molecular weight 127,000, which could be a component of the intestinal transport system(s) responsible for the uptake of orally effective cephalosporins and dipeptides.     

Demonstration of the functional role of conserved Glu-Arg residues in the Staphylococcus aureus ferrichrome transporter

The features that govern the interaction of ligand binding proteins with membrane permeases of cognate ABC transporters are largely unknown. Using sequence alignments and structural modeling based on the structure of the Escherichia coli BtuCD vitamin B₁₂ transporter, we identified six conserved basic residues in the permease, comprised of FhuB and FhuG proteins, in the ferrichrome transporter of Staphylococcus aureus. Using alanine-scanning mutagenesis we demonstrate that two of these residues, FhuB Arg-71 and FhuG Arg-61, play a more dominant role in transporter function than FhuB Arg-74 and Arg-311, and FhuG Arg-64 and Lys-306. Moreover, we show that at positions 71 and 61 in FhuB and FhuG, respectively, arginine cannot be substituted for lysine without loss of transporter function. Previously, our laboratory demonstrated the importance of conserved acidic residues in the ferrichrome binding protein, FhuD2. Taken together, these results support the hypothesis that Glu-Arg salt bridges are critical for the interaction of the ligand binding protein with the transmembrane domains FhuB and FhuG. This hypothesis was further studied by “charge swapping” experiments whereby we constructed a S. aureus strain expressing FhuD2 with conserved residues Glu-97 and Glu-231 replaced by Arg and FhuB and FhuG with conserved basic residues Arg-71 and Arg-61, respectively, replaced by Glu. A strain containing this combination of substitutions restored partial function to the ferrichrome transporter. The results provide a direct demonstration of the functional importance of conserved basic residues on the extracellular surface of the ferrichrome permease in the Gram-positive bacterium S. aureus.     

In vivo reconstitution of an active siderophore transport system by a binding protein derivative lacking a signal sequence

Transport of iron(III) hydroxamates across the inner membrane ofEscherichia coli depends on a binding protein-dependent transport system composed of the FhuB,C and D proteins. The FhuD protein, which is synthesized as a precursor and exported through the cytoplasmic membrane, represents the periplasmic binding protein of the system, accepting as substrates a number of hydroxamate siderophores and the antibiotic albomycin. A FhuD derivative, carrying an N-terminal His-tag sequence instead of its signal sequence and therefore not exported through the inner membrane, was purified from the cytoplasm. Functional activity, comparable to that of wild-type FhuD, was demonstrated for this His-tag-FhuD in vitro by protease protection experiments in the presence of different substrates, and in vivo by reconstitution of iron transport in afhuD mutant strain. The experimental data demonstrate that the primary sequence of the portion corresponding to the mature FhuD contains all the information required for proper folding of the polypeptide chain into a functional solute-binding protein. Moreover, purification of modified periplasmic proteins from the cytosol may be a useful approach for recovery of many polypeptides which are normally exported across the inner membrane and can cause toxicity problems when overproduced.     

Inhibition of hexose transport by adenosine derivatives in human erythrocytes

The human erythrocyte membrane carriers for hexoses and nucleosides have several structural features in common. In order to assess functional similarities, the effects of adenosine derivatives on hexose transport and cytochalasin B binding sites were studied. Adenosine inhibited zero-trans uptake of 3-O-methylglucose half-maximally at 5 mM, while more hydrophobic adenosine deaminase-resistant derivatives were ten- to 20-fold more potent transport inhibitors. However, degradation of adenosine accounted for very little of this difference in potency. Hexose transport was rapidly inhibited by N6-(L-2-phenylisopropyl)adenosine at 5 degrees C in a dose-dependent fashion (EC50 = 240 microM), to lower the transport Vmax without affecting the Km. A direct interaction with the carrier protein was further indicated by the finding that N6-(L-2-phenylisopropyl)adenosine competitively inhibited [3H]cytochalasin B binding to erythrocytes (Ki = 143 microM) and decreased [3H]cytochalasin B photolabeling of hexose carriers in erythrocyte ghosts. The cross-reactivity of adenosine and several of its derivatives with the hexose carrier suggests further homologies between the carriers for hexoses and nucleosides, possibly related to their ability to transport hydrophilic molecules through the lipid core of the plasma membrane.     

Reconstitution of an active lactose carrier in vivo by simultaneous synthesis of two complementary protein fragments.

Escherichia coli lactose permease mediates the proton-driven translocation of galactosides across the cytoplasmic membrane. To define regions important for membrane insertion as well as for biological function, we constructed plasmids encoding different portions of the lactose carrier. Among several lacY deletions, two were obtained that encoded mutant proteins with complementary amino acid sequences. The truncated polypeptide Y71/1 (amino acid residues 1 to 71) comprises the first two alpha-helices predicted for the intact protein, and polypeptide delta Y4-69 carries an internal deletion of this region. Regulated coexpression of these lacY-DNA segments governed by separate but identical lacOP control regions resulted in functional complementation with the following characteristics. (i) Simultaneous synthesis of both incomplete proteins restored transport activity in transport-negative cells, measured as accumulation of [14C]lactose. (ii) Under complementing conditions, but not in the absence of the smaller N-terminal protein, specific radiolabeling of the larger polypeptide by N-ethylmaleimide was prevented by substrate. (iii) The presence of the complementing N-terminal polypeptide was also required for the detection of the larger C-terminal protein by antibodies directed against the C terminus of lactose permease, indicating a stabilizing effect contributed by the smaller N-terminal fragment. Thus, coexpression of lacY mutant genes encoding two nonoverlapping portions of the lactose carrier resulted in reconstitution of a two-subunit protein in the cytoplasmic membrane exhibiting biological properties of intact lactose permease.     

Cross‐species chimeras reveal BamA POTRA and β‐barrel domains must be fine‐tuned for efficient OMP insertion

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram‐negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of β‐barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded β‐barrel precursors via the five polypeptide transport‐associated (POTRA) domains at its N‐terminus. The C‐terminus of BamA folds into a β‐barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram‐negative bacteria and appear to function in a species‐specific manner. Here we investigate the nature of this species‐specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the β‐barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the β‐barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the β‐barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.     

Ferrichrome transport in Escherichia coli K-12: altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the integral membrane protein FhuB.

FhuD is the periplasmic binding protein of the ferric hydroxamate transport system of Escherichia coli. FhuD was isolated and purified as a His-tag-labeled derivative on a Ni-chelate resin. The dissociation constants for ferric hydroxamates were estimated from the concentration-dependent decrease in the intrinsic fluorescence intensity of His-tag-FhuD and were found to be 0.4 microM for ferric aerobactin, 1.0 microM for ferrichrome, 0.3 microM for ferric coprogen, and 5.4 microM for the antibiotic albomycin. Ferrichrome A, ferrioxamine B, and ferrioxamine E, which are poorly taken up via the Fhu system, displayed dissociation constants of 79, 36, and 42 microM, respectively. These are the first estimated dissociation constants reported for a binding protein of a microbial iron transport system. Mutants impaired in the interaction of ferric hydroxamates with FhuD were isolated. One mutated FhuD, with a W-to-L mutation at position 68 [FhuD(W68L)], differed from wild-type FhuD in transport activity in that ferric coprogen supported promotion of growth of the mutant on iron-limited medium, while ferrichrome was nearly inactive. The dissociation constants of ferric hydroxamates were higher for FhuD(W68L) than for wild-type FhuD and lower for ferric coprogen (2.2 microM) than for ferrichrome (156 microM). Another mutated FhuD, FhuD(A150S, P175L), showed a weak response to ferrichrome and albomycin and exhibited dissociation constants two- to threefold higher than that of wild-type FhuD. Interaction of FhuD with the cytoplasmic membrane transport protein FhuB was studied by determining protection of FhuB degradation by trypsin and proteinase K and by cross-linking experiments. His-tag-FhuD and His-tag-FhuD loaded with aerobactin specifically prevented degradation of FhuB and were cross-linked to FhuB. FhuD loaded with substrate and also FhuD free of substrate were able to interact with FhuB.     

Selective Inhibition of Active Uptake of Sucrose into Plasma Membrane Vesicles by Polyclonal Sera Directed against a 42 Kilodalton Plasma Membrane Polypeptide

Several polyclonal sera were raised in rabbits and in mice against putative sucrose carrier proteins, i.e. a 42 kilodalton (O Gallet, R Lemoine, C Larsson, S Delrot [1989] Biochim Biophys Acta 978: 56-64) and a 62 kD (KG Ripp, PV Viitanen, WD Hitz, VR Fransceschi [1988] Plant Physiol 88: 1435-1445) polypeptide of the plasma membrane. The effects of these sera on the active uptake of sucrose and of valine into purified plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves and roots were studied. At a dilution of 1/50, the anti-42 kilodalton sera consistently inhibited sucrose uptake in plasma membranes from leaves or from roots. They had no effect on valine uptake. Under the same experimental conditions, the anti-62 kilodalton sera had no effect on active uptake of sucrose. The data further support the view that a 42 kilodalton polypeptide is a component of the transport system mediating sucrose uptake across the plasma membrane of plant cells.     

MP38 contains the membrane-embedded domain of the lens fiber gap junction protein MP70

A 70-kDa lens membrane polypeptide (MP70) is a specific component of the fiber gap junctions. The C-terminal portion of MP70 is removed by age-related proteolytic processing, leaving an N-terminal 38-kDa polypeptide (MP38) in the membrane. Membrane association and topology of MP70 and of its processed form MP38 have been studied by hydrophobic labeling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and phenyl isothio[14C]cyanate. Membrane-embedded segments have been identified. They are localized in the N-terminal 30-kDa portion of MP70 and MP38. The C-terminal 40-kDa portion of MP70 appears to be exposed entirely at the cytoplasmic side of the junctional membranes. Hence, potentially poreforming peptide segments in the MP70 molecule are conserved upon age-related processing to MP38.     

Molecular dissection of TatC defines critical regions essential for protein transport and a TatB-TatC contact site

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site-specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins.     

Identification of different transport systems for bile salts in sinusoidal and canalicular membranes of hepatocytes

The preservation of the functional polarity of hepatocytes in liver snips (1 x 2 x 4 mm) was demonstrated by fluorescent microscopic studies using the sodium salt of (N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 beta-amino-7 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid. This fluorescent bile salt derivative is not only taken up by hepatocytes of several cell layers at the surface of the snips but also secreted into bile canaliculi. The intact hepatobiliary transport of bile salts by hepatocytes of liver snips demonstrates that they are a useful system for the investigation of those transcellular transport processes which require the integrity of hepatic structure. Photoaffinity labelling of liver snips with the sodium salt of (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan- 24-oyl)-2-aminoethanesulfonic acid revealed that the bile-salt-binding membrane polypeptides with apparent Mr values of 54,000 and 48,000 are exclusively located in the sinusoidal membrane, whereas a single bile-salt-binding polypeptide with an apparent Mr of 100,000 is located in the bile-canalicular membrane. Photoaffinity labelling of liver snips at 4 degrees C, when transcellular bile-salt transport is insignificant, resulted in the labelling of the two sinusoidal membrane polypeptides and practically no labelling of the polypeptide with an apparent Mr of 100,000. This latter polypeptide was also not labelled when Ca2 deprivation abolished bile secretion completely. These results indicate that the directed hepatobiliary transport of bile salts in hepatocytes is accomplished by transport systems which are different for sinusoidal uptake and canalicular secretion.     

Identification and partial purification of a heart mitochondrial membrane proteinase

Membrane-bound proteinase activity was demonstrated by a solid-phase assay system in both beef heart and rat liver mitochondria. The activity was sensitive to SH reagents and assorted proteinase inhibitors. Although stimulated by nonionic detergents, it became labile when solubilized by detergents. The proteinase activity from heart mitochondria copurified with the ADP:ATP translocator protein. Gel electrophoresis of this preparation revealed the translocator polypeptide as well as a number of minor components. In solubilized mitochondria the ADP:ATP translocator polypeptide slowly disappeared upon standing at 0°C as revealed by polyacrylamide gel electrophoresis under denaturing conditions. The loss of this polypeptide was prevented by addition of proteinase inhibitors as well as the translocator affinity ligand, carboxyatractylate. These observations confirm the presence of an integral membrane proteinase in mitochondria and suggest a structural and enzymatic interaction between the proteinase and the ADP:ATP translocator.Abbreviations PMSF phenylmethanesulfonyl fluoride - TPCK l-1-tosylamido-2-phenylethylchloromethyl ketone - TLCK 1-chloro-3-tosylamido-7-amino-l-2-heptanone - NEM N-ethylmaleimide - PCMBS p-chloromercuriphenylsulfonic acid - SDS sodium dodecyl sulfate - MOPS morpholinopropane sulfonate - [I₅₀] concentration of inhibitor required to give 50% inhibition