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1.
The apparatus responsible for translocation of proteins across bacterial membranes is the conserved SecY complex, consisting of SecY, SecE, and SecG. Prior genetic analysis provided insight into the mechanisms of protein export, as well as the interactions between the component proteins. In particular, the prl suppressor alleles of secE and secY, which allow export of secretory proteins with defective signal sequences, have proven particularly useful. Here, we report the isolation of novel mutations in secE and secY, as well as the phenotypic effects of combinations of prl mutations. These new alleles, as well as previously characterized prl mutations, were analyzed in light of the recently published crystal structure of the archaeal SecY complex. Our results support and expand a model of Prl suppressor activity that proposes that all of the prlA and prlG alleles either destabilize the closed state of the channel or stabilize the open form. These mutants thus allow channel opening to occur without the triggering event of signal sequence binding that is required in a wild-type complex. 相似文献
2.
3.
Determinants of performance in the isocitrate dehydrogenase of Escherichia coli. 总被引:3,自引:3,他引:0
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A. M. Dean A. K. Shiau D. E. Koshland Jr 《Protein science : a publication of the Protein Society》1996,5(2):341-347
The substrate specificity of the NADP-dependent isocitrate dehydrogenase of Escherichia coli was investigated by combining site-directed mutagenesis and utilization of alternative substrates. A comparison of the kinetics of the wild-type enzyme with 2R-malate reveals that the gamma-carboxylate of 2R,3S-isocitrate contributes a factor of 12,000,000 to enzyme performance. Analysis of kinetic data compiled for 10 enzymes and nine different substrates reveals that a factor of 1,650 can be ascribed to the hydrogen bond formed between S113 and the gamma-carboxylate of bound isocitrate, a factor of 150 to the negative charge of the gamma-carboxylate, and a factor of 50 for the gamma-methyl. These results are entirely consistent with X-ray structures of Michaelis complexes that show a hydrogen bond positions the gamma-carboxylate of isocitrate so that a salt bridge can form to the nicotinamide ring of NADP. 相似文献
4.
SecY and integral membrane components of the Escherichia coli protein translocation system 总被引:5,自引:0,他引:5
Koreaki Ito 《Molecular microbiology》1992,6(17):2423-2428
Genetic approaches can address the question of how integral membrane Sec factors interact with each other and facilitate protein translocation across the cytoplasmic membrane of E. coli. This review summarizes genetic analyses of SecY, SecE and some other protein translocation factors, utilizing 'prl' mutations, 'sec' mutations, 'suppressor-directed inactivation', 'Sec titration', dominant negative mutations and their suppressors. Evidence suggests that co-ordinate participation of SecY, SecE, SecD, SecF, and probably some other factors, is crucial for the process. 相似文献
5.
In vivo cross-linking of the SecA and SecY subunits of the Escherichia coli preprotein translocase. 总被引:1,自引:0,他引:1
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Precursor protein translocation across the Escherichia coli inner membrane is mediated by the translocase, which is composed of a heterotrimeric integral membrane protein complex with SecY, SecE, and SecG as subunits and peripherally bound SecA. Cross-linking experiments were conducted to study which proteins are associated with SecA in vivo. Formaldehyde treatment of intact cells results in the specific cross-linking of SecA to SecY. Concurrently with the increased membrane association of SecA, an elevated amount of cross-linked product was obtained in cells harboring overproduced SecYEG complex. Cross-linked SecA copurified with hexahistidine-tagged SecY and not with SecE. The data indicate that SecA and SecY coexist as a stable complex in the cytoplasmic membrane in vivo. 相似文献
6.
7.
Microtubular and lamellate membrane structures were observed at the log phase of growth in the stable L-forms of Escherichia coli cultured in the absence of penicillin. 相似文献
8.
SecY is an indispensable component of the protein secretory machinery of Escherichia coli 总被引:8,自引:0,他引:8
K Nishiyama Y Kabuyama J Akimaru S Matsuyama H Tokuda S Mizushima 《Biochimica et biophysica acta》1991,1065(1):89-97
Using a reconstitution system for protein translocation, the involvement of SecY in the translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli was studied. Anti-SecY antibodies raised against the N- and C-terminal sequences prevented the functional reconstitution of the translocation system. Depletion of SecY from the solubilized membrane preparation was performed by treatment with anti-SecY IgG, followed by removal of IgG with protein A-agarose. The SecY-depleted preparation was inactive as to functional reconstitution. However, reconstitution with it was demonstrated in the presence of a protein fraction, which was released from the anti-SecY immunoprecipitate upon addition of the SecY fragment used to raise the antibody. Reconstitution with the SecY-depleted membrane fraction was also demonstrated in the presence of a purified SecY preparation. OmpT proteinase specifically cleaved SecY in the solubilized membrane preparation. The cleavage was accompanied by a decrease in the reconstituted activity. Based on these findings we conclude that SecY is an indispensable component of the secretory machinery. 相似文献
9.
SecE-dependent overproduction of SecY in Escherichia coli. Evidence for interaction between two components of the secretory machinery 总被引:20,自引:0,他引:20
The secY and secE genes were individually cloned and placed under the control of the tac promoter on plasmids. Induction with isopropyl-beta-D-thiogalactopyranoside resulted in the overproduction of SecE, but not that of SecY. The simultaneous induced expression of both genes in the same cells resulted in the overproduction of SecY together with that of SecE. SecY and SecE thus overproduced were localized in the cytoplasmic membrane as those expressed at the normal levels were. It is suggested that SecY and SecE interact with each other in the cytoplasmic membrane. The numbers of the SecY and SecE molecules per cell were estimated. 相似文献
10.
Roles of the C-terminal end of SecY in protein translocation and viability of Escherichia coli
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SecY, a central component of the membrane-embedded sector of protein translocase, contains six cytosolic domains. Here, we examined the importance of the C-terminal cytosolic region of SecY by systematically shortening the C-terminal end and examining the functional consequences of these mutations in vivo and in vitro. It was indicated that the C-terminal five residues are dispensable without any appreciable functional defects in SecY. Mutants missing the C-terminal six to seven residues were partially compromised, especially at low temperature or in the absence of SecG. In vitro analyses indicated that the initial phase of the translocation reaction, in which the signal sequence region of the preprotein is inserted into the membrane, was affected by the lack of the C-terminal residues. SecA binding was normal, but SecA insertion in response to ATP and a preprotein was impaired. It is suggested that the C-terminal SecY residues are required for SecA-dependent translocation initiation. 相似文献
11.
SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase. 总被引:8,自引:0,他引:8
L Brundage C J Fimmel S Mizushima W Wickner 《The Journal of biological chemistry》1992,267(6):4166-4170
The preprotein translocase of Escherichia coli is a multisubunit enzyme with two domains, the peripheral membrane protein SecA and the membrane-embedded SecY/E protein. SecY/E has been isolated as a complex of three polypeptides, SecY, SecE, and band 1. We now present four lines of evidence that the active species of SecY/E is composed of a tightly associated complex of these three subunits: 1) antibodies to SecY efficiently precipitate SecY/E activity as well as all three polypeptides; 2) the proportions of SecY, SecE, and band 1 in the immunoprecipitates are the same as in the starting fraction; 3) the immunoprecipitable complex is not disrupted by treatment with either high salt or urea but is disrupted by brief incubation at 20 degrees C, and the kinetics of dissociation of both band 1 and SecE from SecY at 20 degrees C parallel the loss of translocation ATPase activity; 4) upon immunoprecipitation of similar units of activity of translocase from detergent solutions from either wild-type membranes or a SecY and SecE overproducer strain, the SecE and band 1 subunits are recovered in the same proportions. These data establish that the subunits of SecY/E are firmly associated and that it is the associated complex which is active for translocation. 相似文献
12.
Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. 总被引:50,自引:9,他引:41
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The secY (prlA) gene product is an essential component of the Escherichia coli cytoplasmic membrane, and its function is required for the translocation of exocytoplasmic proteins across the membrane. We have analyzed the orientation of the SecY protein in the membrane by examining the hydropathic character of its amino acid sequence, by testing its susceptibility to proteases added to each side of the membrane, and by characterizing SecY-PhoA (alkaline phosphatase) hybrid proteins constructed by TnphoA transpositions. The orientation of the PhoA portion of the hybrid protein with respect to the membrane was inferred from its enzymatic activity as well as sensitivity to external proteases. The results suggest that SecY contains 10 transmembrane segments, five periplasmically exposed parts, and six cytoplasmic regions including the amino- and carboxyterminal regions. 相似文献
13.
SecY variants that interfere with Escherichia coli protein export in the presence of normal secY 总被引:5,自引:0,他引:5
Takashi Shimoike Yoshinori Akiyama Tadashi Baba Tetsuya Taura Koreaki Ito 《Molecular microbiology》1992,6(9):1205-1210
As an approach for studying how SecY, an integral membrane protein translocation factor of Escherichia coli, interacts with other protein molecules, we isolated a dominant negative mutation, secY-d1, of the gene carried on a plasmid. The mutant plasmid severely inhibited export of maltose-binding protein and less severely of OmpA, when introduced into sec+ cells. It inhibited growth of secY and secE mutant cells, but not of secA and secD mutant cells or wild-type cells. The mutation deletes three amino acids that should be located at the interface of cytoplasmic domain 5 and transmembrane segment 9. We also found that some SecY-PhoA fusion proteins that lacked carboxy-terminal portions of SecY but retain a region from periplasmic domain 3 to transmembrane segment 7 were inhibitory to protein export. We suggest that these SecY variants are severely defective in catalytic function of SecY, which requires cytoplasmic domain 5 and its carboxy-terminal side, but retain the ability to associate with other molecules of the protein export machinery, which requires the central portion of SecY; they probably exert the 'dominant negative' effects by competing with normal SecY for the formation of active Sec complex. These observations should provide a basis for further genetic analysis of the Sec protein complex in the membrane. 相似文献
14.
A well-characterized protein phosphorelay mediates Escherichia coli chemotaxis towards the amino acid attractant aspartate. The protein CheY shuttles between flagellar motors and methyl-accepting chemoreceptor (MCP) complexes containing the linker CheW and the kinase CheA. CheA-CheY phosphotransfer generates phospho-CheY, CheY-P. Aspartate triggers smooth swim responses by inactivation of the CheA bound to the target MCP, Tar; but this mechanism alone cannot explain the observed response sensitivity. Here, we used behavioral analysis of mutants deleted for CheZ, a catalyst of CheY-P dephosphorylation, or the methyltransferase CheR and/or the methylesterase CheB to examine the roles of accelerated CheY-P dephosphorylation and MCP methylation in enhancement of the chemotactic response. The extreme motile bias of the mutants was adjusted towards wild-type values, while preserving much of the aspartate response sensitivity by expressing fragments of the MCP, Tsr, that either activate or inhibit CheA. We then measured responses to small jumps of aspartate, generated by flash photolysis of photo-labile precursors. The stimulus-response relation for Delta cheZ mutants overlapped that for the host strains. Delta cheZ excitation response times increased with stimulus size consistent with formation of an occluded CheA state. Thus, neither CheZ-dependent or independent increases in CheY-P dephosphorylation contribute to the excitation response. In Delta cheB Delta cheR or Delta cheR mutants, the dose for a half-maximal response, [Asp](50), was ca 10 microM; but was elevated to 100 microM in Delta cheB mutants. In addition, the stimulus-response relation for these mutants was linear, consistent with stoichiometric inactivation, in contrast to the non-linear relation for wild-type E. coli. These data suggest that response sensitivity is controlled by differential binding of CheR and/or CheB to distinct MCP signaling conformations. 相似文献
15.
C L Harris 《Journal of bacteriology》1987,169(6):2718-2723
Aminoacyl-tRNA synthetases from several strains of Escherichia coli are shown to elute as a high-molecular-weight complex on 6% agarose columns (Bio-Gel A-5M). In contrast, very little synthetase activity was observed in such complexes on Sephadex G-200 columns, suggesting that these enzymes may interact with or are dissociated during chromatography on dextran. The size of the complex observed on Bio-Gel A-5M was influenced by the method of cell breakage and the salt concentrations present in buffers. The largest complexes (greater than 1,000,000 daltons) were seen with cells broken with a freeze press, whereas with sonicated preparations the average size of the complex was about 400,000 daltons. Extraction of synthetases at 0.15 M NaCl, to mimic physiological salt concentrations, also resulted in high-molecular-weight complexes, as demonstrated by both agarose gel filtration and ultracentrifugation analysis. Evidence is presented that dissociation of some synthetases does occur in the presence of higher salt levels (0.4 M NaCl). Partial purification of the synthetase complex on DEAE-Sephacel was accomplished with only minor dissociation of individual synthetases. These data suggest that a complex(es) of aminoacyl-tRNA synthetase does exist in bacterial cells, just as in eucaryotes, and that the complex may have escaped earlier detection due to its fragility during isolation. 相似文献
16.
Chemotactic stimuli in bacteria are sensed by large sensory complexes, or receptor clusters, that consist of tens of thousands of proteins. Receptor clusters appear to play a key role in signal processing, but their structure remains poorly understood. Here we used fluorescent protein fusions to study in vivo formation of the cluster core, which consists of receptors, a kinase CheA and an assisting protein CheW. We show that receptors aggregate through their cytoplasmic domains even in the absence of other chemotaxis proteins. Clustering is further enhanced by the binding of CheW. Surprisingly, we observed that some fragments of CheA bind receptor clusters well in the absence of CheW, although the latter does assist the binding of full-length CheA. The resulting mode of receptor cluster formation is consistent with an experimentally observed flexible stoichiometry of chemosensory complexes and with assumptions of recently proposed computer models of signal processing in chemotaxis. 相似文献
17.
Summary It has been established that the strain CA8000 of Escherichia coli K 12 produces minicells. This phenotype of CA8000 has been shown to be suppressed by additional mutations in cya or crp genes. Minicell production by cya
+
crp
+
min bacteria is probably a consequence of error, introduced by horizontal growth, in the selection of site on the envelope for initiation of hemispherical growth. 相似文献
18.
A protease complex in the Escherichia coli plasma membrane: HflKC (HflA) forms a complex with FtsH (HflB), regulating its proteolytic activity against SecY. 总被引:8,自引:1,他引:7
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Escherichia coli FtsH (HflB), a membrane-bound ATPase is required for proteolytic degradation of uncomplexed forms of the protein translocase SecY subunit. We have now isolated SecY-stabilizing mutations that cause an amino acid substitution in the HflK-HflC membrane protein complex. Although HflKC protein was believed to have a proteolytic activity against lambda cII protein, deletion of hflK-hflC did not stabilize SecY. Instead, the mutant alleles were partially dominant and overexpression of ftsH suppressed the mutational effects, suggesting that the mutant proteins antagonized the degradation of SecY. These results raise the possibility that even the wild-type HflKC protein acts to antagonize FtsH. Consistent with this notion, the hflkC null mutation accelerated degradation of the SecY24 protein. Furthermore cross-linking, co-immunoprecipitation, histidine-tagging and gel filtration experiments all indicated that FtsH and HflKC form a complex in vivo and in vitro. Finally, purified HflKC protein inhibited the SecY-degrading activity of purified FtsH protein in vitro. These results indicate that the proteolytic activity of FtsH is modulated negatively by its association with HflKC. 相似文献
19.
Qiang Guo Sen Mei Chong Xie Hao Mi Yang Jiang Shi-Ding Zhang Tian-Wei Tan Li-Hai Fan 《Biotechnology and bioengineering》2020,117(6):1738-1746
In the initial step of sugar metabolism, sugar-specific transporters play a decisive role in the passage of sugars through plasma membranes into cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. The present work shows that permeabilized SecY channels can be used as nonspecific sugar transporters in Escherichia coli. SecY with the plug domain deleted allowed the passage of glucose, fructose, mannose, xylose, and arabinose, and, with additional pore-ring mutations, facilitated lactose transport, indicating that sugar passage via permeabilized SecY was independent of sugar stereospecificity. The engineered E. coli showed rapid growth on a wide spectrum of monosaccharides and benefited from the elimination of transport saturation, improvement in sugar tolerance, reduction in competitive inhibition, and prevention of carbon catabolite repression, which are usually encountered with native sugar uptake systems. The SecY channel is widespread in prokaryotes, so other bacteria may also be engineered to utilize this system for sugar uptake. The SecY channel thus provides a unique sugar passageway for future development of robust cell factories for biotechnological applications. 相似文献
20.
Skp of Escherichia coli (OmpH of Salmonella typhimurium) is a protein whose precise function has been obscured by its ubiquity in a wide range of subcellular fractions such as those containing DNA, ribosomes, and outer membranes. Combining in vitro and in vivo techniques we show that Skp is synthesized as a larger precursor that is processed upon translocation across the plasma membrane. Translocation is dependent on the H(+)-gradient, ATP, SecA, and SecY. Upon cellular subfractionation (avoiding non-specific electrostatic interactions) Skp partitions with beta-lactamase into the fraction of soluble, periplasmic proteins. In the context of the export factor properties of Skp previously demonstrated in vitro it is conceivable that this protein is involved in the later steps of protein translocation across the plasma membrane and/or sorting to the outer membrane. 相似文献