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1.
An important role of protein ADP-ribosylation in bacterial morphogenesis has been proposed (J. Bacteriol. 178, 3785-3790; 178, 4935-4941). To clarify the detail of ADP-ribosylation, we identified a new kind of target protein for ADP-ribosylation in Streptomyces coelicolor A3(2) grown to the late growth phase. All four proteins (MalE, BldKB, a periplasmic protein for binding branched-chain amino-acids, and a periplasmic solute binding protein) were functionally similar and participated in the regulation of transport of metabolites or nutrients through the membrane. ADP-ribosylation was likely to occur on a cysteine residue, because the modification group was removed by mercuric chloride treatment. The modification site may be the site of lipoprotein modification necessary for protein export. This report is the first suggesting that certain proteins involved in membrane transport can be ADP-ribosylated.  相似文献   

2.
The histidine permease of Salmonella typhimurium consists of four protein components, one located in the periplasm and three in the cytoplasmic membrane. Genetic evidence indicated that the periplasmic protein interacts with the membrane proteins during transport. We have utilized two different methods to demonstrate that the periplasmic protein cross-links specifically to one of the membrane components, the Q protein. Formaldehyde, a water-soluble permeant molecule was used in vivo. Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate, a photoactivatable cross-linking reagent, was used in vitro in a reconstituted membrane vesicle system. Furthermore, we show that a mutant periplasmic protein, capable of binding substrate but not transporting it, is defective in cross-linking to the membrane protein, indicating this interaction to be a crucial step in the mechanism of transport.  相似文献   

3.
Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD+ to acceptor proteins catalyzed by ADP-ribosyltransferases. Using m-aminophenylboronate affinity chromatography, 2D-gel electrophoresis, in-gel digestion and MALDI-TOF analysis we have identified eight in vitro ADP-ribosylated proteins in Streptomyces coelicolor, which can be classified into three categories: (i) secreted proteins; (ii) metabolic enzymes using NAD+/NADH or NADP+/NADPH as coenzymes; and (iii) other proteins. The secreted proteins could be classified into two functional categories: SCO2008 and SC05477 encode members of the family of periplasmic extracellular solute-binding proteins, and SCO6108 and SC01968 are secreted hydrolases. Dehydrogenases are encoded by SC04824 and SC04771. The other targets are GlnA (glutamine synthetase I., SC02198) and SpaA (starvation-sensing protein encoded by SC07629). SCO2008 protein and GlnA had been identified as ADP-ribosylated proteins in previous studies. With these results we provided experimental support for a previous suggestion that ADP-ribosylation may regulate membrane transport and localization of periplasmic proteins. Since ADP-ribosylation results in inactivation of the target protein, ADP-ribosylation of dehydrogenases might modulate crucial primary metabolic pathways in Streptomyces. Several of the proteins identified here could provide a strong connection between protein ADP-ribosylation and the regulation of morphological differentiation in S. coelicolor.  相似文献   

4.
Bacterial periplasmic binding protein-dependent transport systems require the function of a specific substrate-binding protein, located in the periplasm, and several membrane-bound components. We present evidence for a nucleotide-binding site on one of the membrane components from each of three independent transport systems, the hisP, malK and oppD proteins of the histidine, maltose and oligopeptide permeases, respectively. The amino acid sequence of the oppD protein has been determined and this protein is shown to share extensive homology with the hisP and malK proteins. Three lines of evidence lead us to propose the existence of a nucleotide-binding site on each of these proteins. A consensus nucleotide-binding sequence can be identified in the same relative position in each of the three proteins. The oppD protein binds to a Cibacron Blue affinity column and can be eluted by ATP but not by CTP or NADH. The oppD protein is labelled specifically by the nucleotide affinity analogue 5'-p-fluorosulphonylbenzoyladenosine. The identification of a nucleotide-binding site provides strong evidence that transport by periplasmic binding protein-dependent systems is energized directly by the hydrolysis of ATP or a closely related nucleotide. The hisP, malK and oppD proteins are thus responsible for energy-coupling to their respective transport systems.  相似文献   

5.
The nucleotide sequence of the gene for the spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome was determined. It contained four open reading frames encoding A, B, C, and D proteins. By making several subclones, we showed that expression of all the four proteins was necessary for maximal spermidine and putrescine transport activity. A single transport system was involved in the transport of both spermidine and putrescine. The A protein (Mr 43K) was found to be associated with membranes, as shown by Western blot analysis of the cell fractions. In addition, it had consensus amino acid sequences for the nucleotide binding site. B (Mr 31K) and C (Mr 29K) proteins consisted of six putative transmembrane spanning segments linked by hydrophilic segments of variable length as shown by cell localization of the proteins synthesized in maxicells and by hydropathy profiles. D protein (Mr 39K) was inferred to be a polyamine binding protein existing in a periplasmic fraction from the results of Western blot analysis of the cell fractions and from measurements of polyamine binding to the protein. These results indicate that the spermidine and putrescine transport system can be defined as a bacterial periplasmic transport system.  相似文献   

6.
The ability of rat liver submitochondrial particles to catalyze NAD+ hydrolysis with a transfer of ADP-ribose residues to protein membranes has been demonstrated ADP-ribosylation is directly dependent on NAD+ concentration upon saturation with 1 mM NAD+ and is inhibited by physiological compounds (e.g., ATP, 10 mM; nicotinamide, 10 mM); besides, it is an artificial acceptor of ADP-ribose, arginine methyl ester. It was found that ADP-ribose is accepted by inner mitochondrial membrane protein, whose molecular masses amount to 25-30 kDa. The fact that 5'-AMP is a product of ADP-ribose degradation by snake venom phosphodiesterase suggests that the inner membrane vesiculate proteins are modified by mono(ADP-ribose). Covalent modification of membrane proteins by ADP-ribose leads to citrate transport inhibition in inner membrane vesicles the [14C]citrate uptake is significantly decreased thereby. The ability of ADP-ribosylation inhibitors to restore the citrate transport rate is suggestive of a direct regulatory effect of NAD+-dependent ADP-ribosylation on the activity of citrate-translocating system of inner mitochondrial membranes.  相似文献   

7.
Summary The polyamine content in cells is regulated by both polyamine biosynthesis and its transport. We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) inEscherichia coli. The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system. Furthermore, these two systems were periplasmic transport systems consisting of four kinds of proteins: pPT104 clone encoded potA, -B,-C, and -D proteins and pPT79 clone encoded potF, -G, -H, and -I proteins, judging from the deduced amino acid sequences of the nucleotide sequences of these clones. PotD and -F proteins were periplasmic substrate binding proteins and potA and -G proteins membrane associated proteins having the nucleotide binding site. PotB and -C proteins, and potH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins were similar to each other. The functions of potA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach. In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (potE protein) haveing twelve transmembrane segments, and was active in both the uptake and excretion of putrescine. The uptake was dependent on membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine.  相似文献   

8.
A carrier model in which transport across the cytoplasmic membrane is mediated by a periplasmic binding protein (Krupka, R.M. (1992) Biochim. Biophys. Acta 1110, 1-10) is shown to account for many of the properties of these systems: (i) Michaelis-Menten kinetics; (ii) seemingly irreversible uptake; (iii) the absence of exchange transport and counter-transport; (iv) substrate half-saturation constants that in different systems may be lower or higher than the dissociation constant of the binding protein; (v) the high concentration of the binding protein in the periplasm and its weak association with the membrane component. The binding protein appears to function as a valve or rectifier that permits the substrate to enter the cell, but blocks exit in both the energized and de-energized states. The asymmetry depends on both the abruptness and the extent of the conformational change in the binding protein. Characteristically, these systems build up steep gradients across the membrane, circumstances in which such a valve might be important. In agreement with the mechanism, (a) the binding protein is missing in members of the same family of transporters that function in export of the substrate rather than import; and (b) in Gram-positive organisms, which have no periplasmic space, binding proteins function while anchored to the cytoplasmic membrane.  相似文献   

9.
Although TauA shares few common characteristics with other known periplasmic binding protein, TauA is a putative periplasmic binding protein, part of tauABCD gene cluster involved in sulfonate transport in sulphate starvation condition. This protein was expressed in E. coli BL 21 and purified before to assess its binding functionalities. Measurement of K d value (mean 11.3 nM) by binding/dialysis studies revealed high affinity and specificity with taurine and also indicated that TauA possessed a unique binding site for its ligand. Comparisons with other periplasmic binding proteins suggests TauA plays a major role in ABC transport system and could be ideal candidate to serve as taurine catcher in biological fluids.  相似文献   

10.
Cells of Escherichia coli take up vitamin B(12) (cyano-cobalamin [CN-Cbl]) and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in SALMONELLA: The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His(6) tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.  相似文献   

11.
Agrobacterium tumefaciens VirD4 is essential for DNA transfer to plants. VirD4 presumably functions as a coupling factor that facilitates communication between a substrate and the transport pore. To serve as a coupling protein, VirD4 may be required to localize near the transport apparatus. In a previous study, we observed that several constituents of the transport apparatus localize to the cell membranes. In this study, we demonstrate that VirD4 has a unique cellular location. In immunofluorescence microscopy, cells probed with anti-VirD4 antibodies had foci of fluorescence primarily at the cell poles, indicating that VirD4 localizes to the cell pole. Polar location of VirD4 was not dependent on T-DNA processing, the formation of the transport apparatus and the presence of other Vir proteins. VirD4 is an integral membrane protein with one periplasmic domain. The large cytoplasmic region contains a nucleotide-binding domain. To investigate the role of these domains in DNA transfer, we introduced mutations in virD4 and studied the effect of a mutation on substrate transfer. A deletion of most of the periplasmic domain as well as the alterations of glycine 151 to serine and lysine 152 to alanine led to the complete loss of DNA transfer, indicating that both domains are essential for substrate transfer. Subcellular localization of the mutant proteins indicated that both the periplasmic and the nucleotide-binding domains are required for polar localization of VirD4. The periplasmic domain mutant VirD4Delta36-61 was distributed throughout the cell membrane, whereas the nucleotide binding site mutant VirD4G151S localized to sites other than the cell poles. Polar location of VirD4 suggests a role for the cell pole in DNA transfer.  相似文献   

12.
Periplasmic binding protein-dependent transport systems are composed of a periplasmic substrate-binding protein, a set of 2 (sometimes 1) very hydrophobic integral membrane proteins, and 1 (sometimes 2) hydrophilic peripheral membrane protein that binds and hydrolyzes ATP. These systems are members of the superfamily of ABC transporters. We performed a molecular phylogenetic analysis of the sequences of 70 hydrophobic membrane proteins of these transport systems in order to investigate their evolutionary history. Proteins were grouped into 8 clusters. Within each cluster, protein sequences displayed significant similarities, suggesting that they derive from a common ancestor. Most clusters contained proteins from systems transporting analogous substrates such as monosaccharides, oligopeptides, or hydrophobic amino acids, but this was not a general rule. Proteins from diverse bacteria are found within each cluster, suggesting that the ancestors of current clusters were present before the divergence of bacterial groups. The phylogenetic trees computed for hydrophobic membrane proteins of these permeases are similar to those described for the periplasmic substrate-binding proteins. This result suggests that the genetic regions encoding binding protein-dependent permeases evolved as whole units. Based on the results of the classification of the proteins and on the reconstructed phylogenetic trees, we propose an evolutionary scheme for periplasmic permeases. According to this model, it is probable that these transport systems derive from an ancestral system having only 1 hydrophobic membrane protein.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

13.
Periplasmic transport systems consist of a membrane-bound complex and a periplasmic substrate-binding protein and are postulated to function by translocating the substrate either through a nonspecific pore or through specific binding sites located in the membrane complex. We have isolated mutants carrying mutations in one of the membrane-bound components of the histidine permease of Salmonella typhimurium that allow transport in the absence of both histidine-binding proteins HisJ and LAO (lysine-, arginine-, ornithine-binding protein). All of the mutations are located in a limited region of the nucleotide-binding component of the histidine permease, HisP. The mutants transported substrate in the absence of binding proteins only when the membrane-bound complex was produced in large amounts. At low (chromosomal) levels, the mutant complex was unable to transport substrate in the absence of binding proteins but transported it efficiently in the presence of HisJ. The alterations responsible for the mutations were identified by DNA sequencing; they are closely related to a group of hisP mutations isolated as suppressors of HisJ interaction mutations (G. F.-L. Ames and E. N. Spudich, Proc. Natl. Acad. Sci. USA 73:1877-1881, 1976). The hisP suppressor mutations behaved similarly to these newly isolated mutations despite the entirely different selection procedure. The results are consistent with the HisP protein carrying or contributing to the existence of a substrate-binding site that can be mutated to function in the absence of a binding protein.  相似文献   

14.
A kinetic model for binding protein-mediated arabinose transport.   总被引:2,自引:2,他引:0       下载免费PDF全文
A kinetic model is presented based on the simplest plausible mechanism for bacterial binding protein-dependent transport. The transport phenotypes of the 18 variant arabinose-binding proteins analyzed by Kehres and Hogg (1992, Protein Sci. 1, 1652-1660) (wild type and 17 mutants) are interpreted to mean that in wild-type arabinose uptake the forward transport rate (k(for)) greatly exceeds the dissociation rate (kund) of a binding protein docked with the AraG:AraH membrane complex, and that k(for) dominance is preserved in all of the binding protein surface mutants. The assumptions and predictions of the model are consistent with existing data from other periplasmic transport systems.  相似文献   

15.
Membrane-associated gene products of shock-sensitive bacterial transport operons are often difficult to detect. A 4.5-kilobase DNA fragment, known to completely encode the Salmonella typhimurium tctI operon, was cloned in both orientations behind the T7 phage promoter phi 10 and expressed by using the T7 polymerase-promoter system of Tabor and Richardson (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078, 1985). Under these conditions, five proteins were clearly demonstrated. One DNA strand was shown to encode the periplasmic (29,000-Mr) C protein (as a 31,000-Mr precursor), a 19,000-Mr protein, and a 40,000- to 45,000-Mr protein which ran as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The opposite strand carried the information for two additional proteins of 29,000 and 14,000 Mr. By Tn5 mutagenesis, subcloning of Tn5 insertions, and subcloning of various deletion mutants it was shown that the tctI system is divergently transcribed. The periplasmic binding protein (C protein) is the first product of one operon, followed by the 19,000-Mr and 45,000-Mr integral inner membrane proteins. On the opposite strand only the 29,000-Mr protein was essential for tctI function, and it was found to be weakly attached to the inner membrane. Thus tctI encodes four proteins, one periplasmic, two integral, and one peripheral to the cytoplasmic membrane, with the genes arranged as tctA tctB tctC tctD.  相似文献   

16.
In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787–1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.  相似文献   

17.
Bacteria have evolved several transport mechanisms to maintain metal homeostasis and to detoxify the cell. One mechanism involves an RND (resistance-nodulation-cell division protein family)-driven tripartite protein complex to extrude a variety of toxic substrates to the extracellular milieu. These efflux systems are comprised of a central RND proton-substrate antiporter, a membrane fusion protein, and an outer membrane factor. The mechanism of substrate binding and subsequent efflux has yet to be elucidated. However, the resolution of recent protein crystal structures and genetic analyses of the components of the heavy-metal efflux family of RND proteins have allowed the developments of proposals for a substrate transport pathway. Here two models of substrate extrusion through RND protein complexes of the heavy-metal efflux protein family are described. The funnel model involves the shuttling of periplasmic substrate from the membrane fusion protein to the RND transporter and further on through the outer membrane factor to the extracellular space. Conversely, the switch model requires substrate binding to the membrane fusion protein, inducing a conformational change and creating an open-access state of the tripartite protein complex. The extrusion of periplasmic substrate bypasses the membrane fusion protein, enters the RND-transporter directly via its substrate-binding site, and is ultimately eliminated through the outer membrane channel. Evidence for and against the two models is described, and we propose that current data favor the switch model.  相似文献   

18.
Mutants constitutive for the novel outer membrane protein Ic (e or E) contained a recently discovered binding protein for sn-glycerol-3-phosphate. The corresponding parental strains missing the outer membrane protein Ic (e, E) were negative or strongly reduced in the synthesis of the binding protein. In addition, strains that were previously isolated as mutants constitutive for the sn-glycerol-3-phosphate transport system (ugp+ mutants) and that produced the novel periplasmic proteins GP1 to GP4 also synthesized a new outer membrane protein with the same electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as protein Ic. Screening of different ugp+ mutants revealed the existence of three types in respect to the four novel periplasmic proteins GP1, -2, -3, and -4: (i) one containing all four proteins; (ii) one containing only proteins GP1, -2, and -3; (iii) one containing only proteins GP1, -2, and -4. In confirmation of the data presented in the accompanying paper by Tommassen and Lugtenberg (J. Bacteriol. 143:151–157, 1980), we found that purified GP1 is identical to alkaline phosphatase, whereas purified GP3 has binding activity of inorganic phosphate and is identical to the phosphate-binding protein. Moreover, growth conditions that lead in a wild-type strain to the derepression of alkaline phosphatase synthesis also derepressed the synthesis of the sn-glycerol-3-phosphate-binding protein as well as the corresponding transport system. Thus, the new sn-glycerol-3-phosphate transport system is part of the alkaline phosphatase regulatory system.  相似文献   

19.
Gram-negative bacteria are surrounded by two membranes. In these bacteria, a class of high affinity transport systems for concentrating substrates from the medium into the cell, involves a binding protein located between the outer and inner membranes, in the periplasmic region. These 'periplasmic binding-proteins' are thought to bind the substrate in the vicinity of the inner membrane, and to transfer it to a complex of inner membrane proteins for concentration into the cytoplasm. We report evidence leading us to propose that a Gram-positive bacterium, Streptococcus pneumoniae, and a mycoplasma, Mycoplasma hyorhinis, which are surrounded by a single membrane and have therefore no periplasmic region, possess an equivalent to the high affinity periplasmic binding-protein dependent transport systems, i.e. extra-cytoplasmic binding lipoprotein dependent transport systems. The 'binding lipoproteins' would be maintained at proximity of the inner membrane by insertion of their N-terminal glyceride-cysteine into this membrane.  相似文献   

20.
The alpha subunits of Gi (Gi alpha) and Gs (guanine-nucleotide-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in differentiated HL-60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N-formylmethionine). The ADP-ribosylation site of Gi alpha modified by cholera toxin appeared to be different from that modified by pertussis toxin [Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M. & Katada, T. (1989) J. Biol. Chem. 264, 21,394-21,400]. This allowed us to investigate how the two types of ADP-ribosylation influence the function of the signal-coupling protein. The major findings observed in HL-60 cell membranes, where the same Gi alpha molecule was ADP-ribosylated by treatment of the membranes with either toxin, are summarized as follows. (a) More fMLF bound with a high affinity to cholera-toxin-treated membranes than to the control membranes. The high-affinity binding was, however, not observed in pertussis-toxin-treated membranes. (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis-toxin-treated membranes. In contrast, fMLF-dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera-toxin-treated membranes. (c) Gi alpha, once modified by cholera toxin, still served as a substrate of pertussis-toxin-catalyzed ADP-ribosylation; however, the ADP-ribosylation rate of modified Gi was much lower than that of intact Gi. These results suggested that Gi ADP-ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high-affinity fMLF receptors, and that hydrolysis of GTP bound to the alpha subunit was selectively impaired by its ADP-ribosylation by cholera toxin. Thus, unlike the ADP-ribosylation of Gi by pertussis toxin, cholera-toxin-induced modification would be of great advantage to the interaction of Gi with receptors and effectors that are regulated by the signal-coupling protein. This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal-transduction systems.  相似文献   

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