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1.
Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains 总被引:7,自引:0,他引:7
Two proteolytic fragments of the Escherichia coli mannitol permease (EIImtl) have been identified on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels and mapped with respect to the membrane. EIImtl was selectively radiolabeled with either [35S]methionine or a mixture of 14C-labeled amino acids in E. coli minicells harboring a plasmid containing the mannitol operon. The intact permease (Mr 65,000) in everted vesicles derived from labeled minicells was cleaved by mild trypsinolysis into two smaller fragments (Mr 34,000 and 29,000). The 34,000-dalton fragment remained in the membrane and was insensitive to further proteolysis by trypsin. This fragment was identified as the N-terminal half of the protein by comparing the amount of the original [35S]methionine label that it retained with the known differential distribution of methionine in the two halves of EIImtl. The 29,000-dalton fragment, which was released into the soluble fraction and was sensitive to further trypsinolysis, therefore corresponds to the C-terminal half of the mannitol permease. Both fragments were shown to be antigenically related to EIImtl by immunoblotting with anti-EIImtl antibody. The 34,000-dalton fragment was further shown to form an oligomer under conditions which allow the intact enzyme to dimerize, suggesting that this domain plays an important role in EIImtl subunit interactions. These results support a model in which EIImtl consists of two domains of approximately equal size: a membrane-bound, N-terminal domain with a tendency to self-associate, and a cytoplasmic C-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
2.
I Yamato 《Journal of biochemistry》1992,111(4):444-450
Lactose permease, the lacY gene product in Escherichia coli, is an integral membrane protein. Its induction was examined in secAts and secYts mutants by measuring o-nitrophenyl-beta-galactoside uptake activity. In contrast to the synthesis of the maltose binding protein, the malE gene product, which is dependent on the secA and secY gene products, lactose permease seemed to be produced and integrated functionally into membrane independently of SecA or SecY. Gene fusion of the lamB signal sequence to the N-terminal part of the lactose permease gene resulted in production of active fused permease in the E. coli membrane. The signal sequence did not seem to be processed, judging from its mobility on SDS polyacrylamide gel electrophoresis. E. coli cell growth was super-sensitive to induction of production of the fused permease with the signal sequence in contrast to induction of the normal lactose permease. These results are consistent with the above observation that production and integration of LacY protein into membrane is relatively independent of the SecY protein that may have a certain specificity for the signal sequence or, more generally, membrane translocation intermediates. 相似文献
3.
Substrate and phospholipid specificity of the purified mannitol permease of Escherichia coli 总被引:1,自引:0,他引:1
G R Jacobson L E Tanney D M Kelly K B Palman S B Corn 《Journal of cellular biochemistry》1983,23(1-4):231-240
D-Mannitol is transported and phosphorylated by a specific enzyme II of the phosphotransferase system of Escherichia coli. This protein was purified previously in detergent solution and has been partially characterized. As one approach in understanding the structure and mechanism of this enzyme/permease, we have tested a number of sugar alcohols and their derivatives as substrates and/or inhibitors of this protein. Our results show that the mannitol permease is highly, but not absolutely, specific for D-mannitol. Compounds accepted by the enzyme include those with substitutions in the C-2(= C-5) position of the carbon backbone of the natural substrate as well as D-mannonic acid, one heptitol and one pentitol. All of these compounds were both inhibitors and substrates for the mannitol permease except for D-mannoheptitol, which was an inhibitor but was not phosphorylated by the enzyme. No compound examined, however, exhibited an affinity for the enzyme as high as that for its natural substrate. We have also investigated the phospholipid requirements of the mannitol permease using phospholipids purified from E coli. The purified protein was significantly activated by phosphatidylethanolamine, but little activation was observed with phosphatidylglycerol or cardiolipin. These observations partially delineate requirements for interaction of sugar alcohols and phospholipids with the mannitol permease. They suggest approaches for the design of specific active site probes for the protein, and strategies for stabilizing the enzyme's activity in vitro. 相似文献
4.
Membrane insertion of the Escherichia coli MalF protein in cells with impaired secretion machinery 总被引:10,自引:0,他引:10
The MalF protein is an integral membrane protein of Escherichia coli containing eight membrane-spanning stretches and a large periplasmic domain of approximately 180 amino acids. We have asked whether this protein is dependent for its membrane insertion on the bacterial secretion machinery specified by the sec genes. Using azide to inhibit the SecA protein and sec mutants to reduce the functioning of the machinery, we have studied the membrane assembly of MalF and beta-galactosidase and alkaline phosphatase fusions to MalF. In no case did we see an effect of reducing sec gene function on the insertion of MalF or fusion proteins. Selection for mutants that would cause internalization of a MalF-beta-galactosidase hybrid protein yielded no mutations in sec genes. Our results suggest that MalF can assemble in the membrane independently of the bacterial secretion machinery. 相似文献
5.
SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane. 总被引:4,自引:2,他引:2
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SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo. 相似文献
6.
The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypoosmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon but requires YidC. Depletion of YidC inhibits translocation of the protein across the membrane. Insertion of MscL occurs primarily in a proton motive force-independent manner. The hydrophilic loop region of MscL has 29 residues that include 5 charged residues. Altering the charges in the periplasmic loop of MscL affects the requirements for membrane insertion. The introduction of one, two or three negatively charged amino acids makes the insertion dependent on the electrochemical membrane potential and gradually dependent on the Sec translocon, whereas the addition of five negatively charged residues as well as the addition of three positively charged residues inhibits membrane insertion of MscL. However, we find that the mutant with three uncharged residues requires both the SecYEG complex and YidC but not SecA for membrane insertion. In vivo cross-linking data showed that the newly synthesized MscL interacts with YidC and with SecY. Therefore, the MscL mutants use a membrane insertion mechanism that involves SecYEG and YidC simultaneously. 相似文献
7.
Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking 总被引:1,自引:0,他引:1
van Montfort BA Schuurman-Wolters GK Wind J Broos J Robillard GT Poolman B 《The Journal of biological chemistry》2002,277(17):14717-14723
A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)(3)-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning alpha-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys(73)). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys(134)), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys(134) becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for alpha-helix packing in the mannitol permease. 相似文献
8.
Membrane topology model of Escherichia coli alpha-ketoglutarate permease by phoA fusion analysis. 总被引:3,自引:0,他引:3
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Escherichia coli alpha-ketoglutarate permease (KgtP) is a 432-amino-acid protein that symports alpha-ketoglutarate and protons. KgtP was predicted to contain 12 membrane-spanning domains on the basis of a calculated hydropathy profile. The membrane topology model of KgtP was analyzed by using kgtP-phoA gene fusions and measuring alkaline phosphatase activities in cells expressing the chimeric proteins. Comparisons of the phosphatase activity levels and the locations of the KgtP-PhoA junctions are consistent with the predicted membrane topology model of KgtP. 相似文献
9.
The GltS Na+/glutamate permease of Escherichia coli is the most extensively studied member of the ESS family of bacterial glutamate:Na+ symporters. This paper presents the membrane topology analysis of the GltS with translational alkaline phosphatase and beta-galactosidase gene fusions generated by TnphoA, nested deletions and targeted fusions. The topology model suggested by the translational fusion technique is compared with the MemGen model and discussed in detail. 相似文献
10.
Subunit and amino acid interactions in the Escherichia coli mannitol permease: a functional complementation study of coexpressed mutant permease proteins.
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Mannitol-specific enzyme II, or mannitol permease, of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system of Escherichia coli carries out the transport and phosphorylation of D-mannitol and is most active as a dimer in the membrane. We recently reported the importance of a glutamate residue at position 257 in the binding and transport of mannitol by this protein (C. Saraceni-Richards and G. R. Jacobson, J. Bacteriol. 179:1135-1142, 1997). Replacing Glu-257 with alanine (E257A) or glutamine (E257Q) eliminated detectable mannitol binding and transport by the permease. In contrast, an E257D mutant protein was able to bind and phosphorylate mannitol in a manner similar to that of the wild-type protein but was severely defective in mannitol uptake. In this study, we have coexpressed proteins containing mutations at position 257 with other inactive permeases containing mutations in each of the three domains of this protein. Activities of any active heterodimers resulting from this coexpression were measured. The results show that various inactive mutant permease proteins can complement proteins containing mutations at position 257. In addition, we show that both Glu at position 257 and His at position 195, both of which are in the membrane-bound C domain of the protein, must be on the same subunit of a permease dimer in order for efficient mannitol phosphorylation and uptake to occur. The results also suggest that mannitol bound to the opposite subunit within a permease heterodimer can be phosphorylated by the subunit containing the E257A mutation (which cannot bind mannitol) and support a model in which there are separate binding sites on each subunit within a permease dimer. Finally, we provide evidence from these studies that high-affinity mannitol binding is necessary for efficient transport by mannitol permease. 相似文献
11.
12.
Insertion of the mannitol permease into the membrane of Escherichia coli. Possible involvement of an N-terminal amphiphilic sequence 总被引:4,自引:0,他引:4
Y Yamada Y Y Chang G A Daniels L F Wu J M Tomich M Yamada M H Saier 《The Journal of biological chemistry》1991,266(27):17863-17871
The in vivo membrane assembly of the mannitol permease, the mannitol Enzyme II (IImtl) of the Escherichia coli phosphotransferase system, has been studied employing molecular genetic approaches. Removal of the N-terminal amphiphilic leader of the permease and replacement with a short hydrophobic sequence resulted in an inactive protein unable to transport mannitol into the cell or catalyze either phosphoenol-pyruvate-dependent or mannitol 1-phosphate-dependent mannitol phosphorylation in vitro. The altered protein (68 kDa) was quantitatively cleaved by an endogenous protease to a membrane-associated 39-kDa fragment and a soluble 28-kDa fragment as revealed by Western blot analyses. Overproduction of the wild-type plasmid-encoded protein also led to cleavage, but repression of the synthesis of the plasmid-encoded enzyme by inclusion of glucose in the growth medium prevented cleavage. Several mtlA-phoA gene fusions encoding fused proteins with N-terminal regions derived from the mannitol permease and C-terminal regions derived from the mature portion of alkaline phosphatase were constructed. In the first fusion protein, F13, the N-terminal 13-aminoacyl residue amphiphilic leader sequence of the mannitol permease replaced the hydrophobic leader sequence of alkaline phosphatase. The resultant fusion protein was inefficiently translocated across the cytoplasmic membrane and became peripherally associated with both the inner and outer membranes, presumably via the noncleavable N-terminal amphiphilic sequence. The second fusion protein, F53, in which the N-terminal 53 residues of the mannitol permease were fused to alkaline phosphatase, was efficiently translocated across the cytoplasmic membrane and was largely found anchored to the inner membrane with the catalytic domain of alkaline phosphatase facing the periplasm. This 53-aminoacyl residue sequence included the amphiphilic leader sequence and a single hydrophobic, potentially transmembrane, segment. Analyses of other MtlA-PhoA fusion proteins led to the suggestion that internal amphiphilic segments may function to facilitate initiation of polypeptide trans-membrane translocation. The dependence of IImtl insertion on the N-terminal amphiphilic leader sequence was substantiated employing site-specific mutagenesis. The N-terminal sequence of the native permease is Met-Ser-Ser-Asp-Ile-Lys-Ile-Lys-Val-Gln-Ser-Phe-Gly.... The following point mutants were isolated, sequenced, and examined regarding the effects of the mutations on insertion of IImtl into the membrane: 1) S3P; 2) D4P; 3) D4L; 4) D4R; 5) D4H; 6) I5N; 7) K6P; and 8) K8P.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
13.
H H Liebke 《Journal of bacteriology》1987,169(3):1174-1181
To define the anti-SecA-LacZ antiserum, immunoprecipitates produced with either whole anti-SecA-LacZ rabbit antiserum or affinity-purified antibodies were used to analyze nondenatured lysates of Escherichia coli. The antiserum contains antibodies that recognize different proteins. Antibody purified by preadsorption to the SecA-LacZ hybrid protein precipitated only the SecA protein from extracts. In contrast, antibody purified from the intact SecA protein precipitated several additional proteins with SecA protein. Ribosomal protein L7L12 is one of the polypeptides coprecipitated with SecA protein by antibody purified by immunoadsorption to the intact SecA protein as well as by unfractionated anti-SecA-LacZ antiserum. Two-dimensional gel electrophoresis of the SecA protein immunoprecipitated by either antiserum or purified antibody indicated that the SecA protein exists in at least two, and probably four, isoforms. Only one of the SecA isoforms is present in a ribosomal preparation. 相似文献
14.
The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on sequence analysis of the putative protein in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm had been proposed (J. Pi, P. J. Wookey, and A. J. Pittard, J. Bacteriol. 173:3622-3629, 1991). This topological model of PheP has been further examined by generating protein fusions with alkaline phosphatase. Twenty-five sandwich fusion proteins have been constructed by inserting the 'phoA gene at specific sites within the pheP gene. In general, the PhoA activities of the fusions support a PheP topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, alterations to the model, affecting spans III and VI, were indicated by this analysis and were supported by additional site-directed mutagenesis of some of the residues involved. 相似文献
15.
Mannose permease of Escherichia coli. Domain structure and function of the phosphorylating subunit 总被引:9,自引:0,他引:9
The mannose permease of Escherichia coli is a component of the phosphotransferase system. It transports mannose and related hexoses by a mechanism that couples sugar transport with sugar phosphorylation. It is a complex consisting of two transmembrane subunits (II-PMan and II-MMan) and a hydrophilic subunit (IIIMan). IIIMan also exists in a soluble form as dimer in the cytoplasm. Each monomer of IIIMan consists of two structurally and functionally distinct domains which are linked by a flexible hinge of the sequence KAAPAPAAAAPKAAPTPAKP. Both domains are transiently phosphorylated. The NH2-terminal domain (P13) is phosphorylated at N-3 of His-10 by the cytoplasmic phosphorylcarrier protein phospho-HPr. The COOH-terminal domain (P20) is phosphorylated by P13 at N-1 of His-175. Phosphoryltransfer occurs not only between P13 and P20 on the same IIIMan subunit but also between isolated domains and between domains on different subunits of the dimer. In the presence of the IIMan subunits, the phosphoryl group is directly transferred from His-175 of P20 to the sugar substrates of the permease. The P13 domain contains the contact sites for dimerization of IIIMan. The P20 domain contains the contact sites for interaction with the IIMan subunits. By reconstructing the ptsL gene, the two domains were expressed as individual polypeptides and the length of the hinge between P13 and P20 was changed. The in vivo and in vitro activities of mutant IIIMan were little affected by these modifications. The hinge is highly sensitive to proteolytic cleavage in vitro and its specificity for proteases can be modified by introducing the appropriate specificity determinants. 相似文献
16.
17.
Some properties of the sulphate transport system and the isolation of sulphate permease mutants in E. coli K12 are described. The gene coding for sulphate permease is located in the same region as the cysA gene in Salmonella typhimurium. 相似文献
18.
A seven-amino-acid cleavage site specific for tobacco etch virus (TEV) protease was introduced into SecA at two separate positions after amino acids 195 and 252. Chromosomal wild-type secA was replaced by these secA constructs. Simultaneous expression of TEV protease led to cleavage of both SecA derivatives. In the functional SecA dimer, proteolysis directly indicated surface exposure of the TEV protease cleavage sites. Cleavage of SecA near residue 195 generated an unstable proteolysis product and a secretion defect, suggesting that this approach could be used to inactivate essential proteins in vivo. 相似文献
19.
SecA protein: autoregulated ATPase catalysing preprotein insertion and translocation across the Escherichia coli inner membrane 总被引:9,自引:1,他引:8
Donald B. Oliver 《Molecular microbiology》1993,7(2):159-165
Recent insight into the biochemical mechanism of protein translocation in Escherichia coli indicates that SecA ATPase is required both for the initial binding of preproteins to the inner membrane as well as subsequent translocation across this structure. SecA appears to promote these events by direct recognition of the preprotein or preprotein-SecB complex, binding to inner-membrane anionic phospholipids, insertion into the membrane biiayer and association with the preprotein translocator, SecY/SecE. ATP binding appears to control the affinity of SecA for the various components of the system and ATP hydrolysis promotes cycling between its different biochemical states. As a component likely to catalyse a rate-determining step in protein secretion, SecA synthesis is co-ordinated with the activity of the protein export pathway. This form of negative reguiation appears to rely on SecA protein binding to its mRNA and repressing translation if conditions of rapid protein secretion prevail within the cell. A precise biochemical scheme for SecA-dependent catalysis of protein export and the details of secA regulation appear to be close at hand. The evolutionary conservation of SecA protein among eubacteria as well as the general requirement for translocation ATPases in other protein secretion systems argues for a mechanistic commonality of all prokaryotic protein export pathways. 相似文献
20.
Truncated forms of Escherichia coli lactose permease: models for study of biosynthesis and membrane insertion. 总被引:7,自引:1,他引:6
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U Stochaj H J Fritz C Heibach M Markgraf A von Schaewen U Sonnewald R Ehring 《Journal of bacteriology》1988,170(6):2639-2645
Using in vitro DNA manipulations, we constructed different lacY alleles encoding mutant proteins of the Escherichia coli lactose carrier. With respect to structural models developed for lactose permease, the truncated polypeptides represent model systems containing approximately one, two, four, and five of the N-terminal membrane-spanning alpha-helices. In addition, a protein carrying a deletion of predicted helices 3 and 4 was obtained. The different proteins were radiolabeled in plasmid-bearing E. coli minicells and were found to be stably integrated into the lipid bilayer. The truncated polypeptides of 50, 71, 143, and 174 N-terminal amino acid residues resembled the wild-type protein in their solubilization characteristics, whereas the mutant protein carrying an internal deletion of amino acid residues 72 to 142 of the lactose carrier behaved differently. Minicell membrane vesicles containing truncated proteins comprising amino acid residues 1 to 143 or 1 to 174 were subjected to limited proteolysis. Upon digestion with proteases of different specificities, the same characteristic fragment that was also produced from the membrane-associated wild-type protein was found to accumulate under these conditions. It has previously been shown to contain the intact N terminus of lactose permease. This supports the idea of an independent folding and membrane insertion of this segment even in the absence of the C-terminal part of the molecule. The results suggest that the N-terminal region of the lactose permease represents a well-defined structural domain. 相似文献