SecE-dependent overproduction of SecY in Escherichia coli. Evidence for interaction between two components of the secretory machinery

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.     

SecA, an essential component of the secretory machinery of Escherichia coli, exists as homodimer.

Size exclusion chromatography of the cytosolic fraction of SecA-overproducing cells of Escherichia coli suggested that SecA, an essential component of the secretory machinery, exists as an oligomer. The subunit structure of SecA was then studied using a purified specimen. Estimation of the molecular mass by means of ultracentrifugation and chemical crosslinking analysis revealed that SecA exists as a homodimer. The purified SecA was denatured in 6 M guanidine-HCl and renatured to a dimer, which was fully active in terms of translocation, even in the presence of 1 mM dithiothreitol. It is suggested that the dimeric structure is not critically maintained by disulfide bonding between the two subunits, each of which contains four cysteine residues.     

Skp is a periplasmic Escherichia coli protein requiring SecA and SecY for export

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.     

SecG is an auxiliary component of the protein export apparatus of Escherichia coli

SecY, SecE and SecG form the membrane-embedded core complex of the Escherichia coli protein export apparatus. These three proteins co-purify and can be co-immunoprecipitated, demonstrating that they are closely associated. While SecE and SecY are generally accepted as essential components of translocase, the role of SecG is more ambiguous. It is commonly believed that deletion of secG causes a cold-sensitive phenotype and a severe defect in export, even though some reports have indicated otherwise. However, we demonstrate that deletion of secG does not produce a cold-sensitive phenotype or a strong export defect in most genetic backgrounds. The more common result is that deletion of secG causes only a mild export defect and does not result in conditional lethality. We propose that the role of SecG is not fundamental to the export process, but is merely auxiliary – as suggested previously by biochemical data – and is physiologically important only when cells are otherwise compromised. Received: 22 July 1999 / Accepted: 11 November 1999     

SecF stabilizes SecD and SecY, components of the protein translocation machinery of the Escherichia coli cytoplasmic membrane.

The effect of the overproduction of SecF encoded by the tac-secF gene on a plasmid on the synthesis of other Sec proteins was studied in Escherichia coli. SecF overproduction resulted in the simultaneous overproduction of SecD encoded by the tac-secD gene on a plasmid. Deletion of the orf6 gene, located downstream of the secF gene, had no effect on SecD overproduction. A pulse-chase experiment revealed that the overproduction was due to stabilization of SecD with SecF. SecF overproduction also resulted in the overproduction of SecY encoded by the tac-secY gene on a plasmid as well. SecF overproduction also enhanced the level of SecY expressed by the chromosomal secY gene. This SecF effect was not due to its effect on SecD or SecE, since SecF overproduction did not affect the levels of SecD and SecE expressed by the chromosomal secD and secE genes, respectively. SecE-dependent overproduction of SecY has already been demonstrated. It is suggested that SecF interacts with both SecD and SecY. SecE-SecY interaction has been demonstrated. It is likely, therefore, that all Sec proteins in the cytoplasmic membrane interact with each other.     

Export of Escherichia coli alkaline phosphatase attached to an integral membrane protein, SecY

The SecY protein is a membrane-bound factor required for bacterial protein export and embedded in the cytoplasmic membrane by its 10 transmembrane segments. We previously proposed a topology model for this protein by adapting the Manoil-Beckwith TnphoA approach, a genetic method to assign local disposition of a membrane protein from the enzymatic activity of the alkaline phosphatase (PhoA) mature sequence attached to the various regions. SecY-PhoA hybrid proteins with the PhoA domain exported to the periplasmic side of the membrane have been obtained at the five putative periplasmic domains of the SecY sequence. We now extended this method to apply it to follow export of the newly synthesized PhoA domain. Trypsin treatment of detergent-solubilized cell extracts digested the internalized (unfolded) PhoA domain but not those exported and correctly folded. One of the hybrid proteins was cleaved in vivo after export to the periplasm, providing a convenient indication for the export. Results of these analyses indicate that export of the PhoA domain attached to different periplasmic regions of SecY occurs rapidly and requires the normal functioning of the secY gene supplied in trans. Thus, this membrane protein with multiple transmembrane segments contains multiple export signals which can promote rapid and secY-dependent export of the PhoA mature sequence attached to the carboxyl-terminal sides.     

Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli.

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.     

SecY and integral membrane components of the Escherichia coli protein translocation system

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.     

A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein

We have shown previously that some particular mutations in bacteriophage Mu repressor, the frameshift vir mutations, made the protein very sensitive to the Escherichia coli ATP-dependent Clp protease. This enzyme is formed by the association between a protease subunit (ClpP) and an ATPase subunit. ClpA, the best characterized of these ATPases, is not required for the degradation of the mutant Mu repressors. Recently, a new potential ClpP associated ATPase, ClpX, has been described. We show here that this new subunit is required for Mu vir repressor degradation. Moreover, ClpX (but not ClpP) was found to be required for normal Mu replication. Thus ClpX has activities that do not require its association with ClpP. In the pathway of Mu replicative transposition, the block resides beyond the strand transfer reaction, i.e. after the transposition reaction per se is completed, suggesting that ClpX is required for the transition to the formation of the active replication complex at one Mu end. This is a new clear-cut case of the versatile activity of polypeptides that form multi-component ATP-dependent proteases.     

SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli.

Bacterial protein export requires two forms of energy input, ATP and the membrane electrochemical potential. Using an in vitro reaction reconstituted with purified soluble and peripheral membrane components, we can now directly measure the translocation-coupled hydrolysis of ATP. This translocation ATPase requires inner membrane vesicles, SecA protein and translocation-competent proOmpA. The stimulatory activity of membrane vesicles can be blocked by either antibody to the SecY protein or by preparing the membranes from a secY-thermosensitive strain which had been incubated at the non-permissive temperature in vivo. The SecA protein itself has more than one ATP binding site. 8-azido-ATP inactivates SecA for proOmpA translocation and for translocation ATPase, yet does not inhibit a low level of ATP hydrolysis inherent in the isolated SecA protein. These data show that the SecA protein has a central role in coupling the hydrolysis of ATP to the transfer of pre-secretory proteins across the membrane.     

Identification of an indispensable amino acid for ppGpp synthesis of Escherichia coli SpoT protein

Amino acid substitutions were introduced into a structurally flexible and highly conserved region of Escherichia coli SpoT protein. SpoT protein changed from Asp to Ala at the 293rd position did not restore cell growth of E. coli CF8295 (delta relA, delta spoT) and did not accumulate ppGpp in the cell, suggesting that the Asp293 is indispensable for ppGpp synthesis of the protein.     

Differential translocation of protein precursors across SecY-deficient membranes of Escherichia coli: SecY is not obligatorily required for translocation of certain secretory proteins in vitro.

SecY, a component of the protein translocation system in Escherichia coli, was depleted at a nonpermissive temperature in a strain which had a temperature-sensitive polar effect on the expression of its secY. Membrane vesicles prepared from these cells, when grown at the nonpermissive temperature, contained about 5% SecY and similarly low levels of SecG. As expected, translocation of alkaline phosphatase precursors across these SecY-deficient membranes was severely impaired and appeared to be directly related to the decrease of SecY amounts. However, despite such a dramatic reduction in SecY and SecG levels, these membranes exhibited 50 to 70% of the wild-type translocation activity, including the processing of the signal peptide, of OmpA precursor (proOmpA). This translocation activity in SecY-deficient membranes was still SecA and ATP dependent and was not unique to proOmpA, as lipoprotein and lambda receptor protein precursors were also transported efficiently. Membranes that were reconstituted from these SecY-depleted membranes contained undetectable amounts of SecY yet were also shown to possess substantial translocation activity for proOmpA. These results indicate that the requirement of SecY for translocation is not obligatory for all secretory proteins and may depend on the nature of precursors. Consequently, it is unlikely that SecY is the essential core channel through which all precursors traverse across membranes; rather, SecY probably contributes to efficiency and specificity.     

Binding of lactoferrin and free secretory component to enterotoxigenic Escherichia coli

To compare the genomic variation of Helicobacter pylori in samples obtained from patients with perforated peptic ulcer, living in the same area of Estonia but belonging to different nationalities, 50 non-consecutive patients (32 Estonians and 18 Russians) admitted in the Tartu University Hospital in 1997-1999 were studied. Gastric samples of antral mucosa were obtained during operation and analysed histologically and with PCR for detection of different genotypes of H. pylori (cagA and vacA s and m subtypes). Among the 50 perforated peptic ulcer patients with histologically proven H. pylori colonisation no sample of gastric mucosa showed the s1b subtype of the vacA gene. The perforated peptic ulcer patients were mainly infected with cagA (82%) and s1 (98%) genotypes of H. pylori. The distribution of s1a/m1, s1a/m2 and s2/m2 subtypes of vacA genes was statistically different in Estonian and Russian patients (P<0.05). In conclusion differences in the distribution of vacA s and m subtypes of H. pylori were revealed between Estonian and Russian patients with perforated peptic ulcer from Southern Estonia.     

RtcB is the RNA ligase component of an Escherichia coli RNA repair operon

RNA 2',3'-cyclic phosphate ends play important roles in RNA metabolism as substrates for RNA ligases during tRNA restriction-repair and tRNA splicing. Diverse bacteria from multiple phyla encode a two-component RNA repair cassette, comprising Pnkp (polynucleotide kinase-phosphatase-ligase) and Hen1 (RNA 3'-terminal ribose 2'-O-methyltransferase), that heals and then seals broken tRNAs with 2',3'-cyclic phosphate and 5'-OH ends. The Pnkp-Hen1 repair operon is absent in the majority of bacterial species, thereby raising the prospect that other RNA repair systems might be extant. A candidate component is RNA 3'-phosphate cyclase, a widely distributed enzyme that transforms RNA 3'-monophosphate termini into 2',3'-cyclic phosphates but cannot seal the ends it produces. Escherichia coli RNA cyclase (RtcA) is encoded in a σ(54)-regulated operon with RtcB, a protein of unknown function. Taking a cue from Pnkp-Hen1, we purified E. coli RtcB and tested it for RNA ligase activity. We report that RtcB per se seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester. We speculate that: (i) RtcB might afford bacteria a means to recover from stress-induced RNA damage; and (ii) RtcB homologs might catalyze tRNA repair or splicing reactions in archaea and eukarya.     

Roles of the C-terminal end of SecY in protein translocation and viability of Escherichia coli

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.     

SecY variants that interfere with Escherichia coli protein export in the presence of normal secY

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.     

Crystal structure of Escherichia coli SufC, an ABC-type ATPase component of the SUF iron-sulfur cluster assembly machinery

SufC is an ATPase component of the SUF machinery, which is involved in the biosynthesis of Fe-S clusters. To gain insight into the function of this protein, we have determined the crystal structure of Escherichia coli SufC at 2.5A resolution. Despite the similarity of the overall structure with ABC-ATPases (nucleotide-binding domains of ABC transporters), some key differences were observed. Glu171, an invariant residue involved in ATP hydrolysis, is rotated away from the nucleotide-binding pocket to form a SufC-specific salt bridge with Lys152. Due to this salt bridge, D-loop that follows Glu171 is flipped out to the molecular surface, which may sterically inhibit the formation of an active dimer. Thus, the salt bridge may play a critical role in regulating ATPase activity and preventing wasteful ATP hydrolysis. Furthermore, SufC has a unique Q-loop structure on its surface, which may form a binding site for its partner proteins, SufB and/or SufD.     

Characterization of a region in mature LamB protein that interacts with a component of the export machinery of Escherichia coli

It has been shown that the synthesis of an export-defective protein can interfere with the normal export process in Escherichia coli by limiting the availability of SecB protein, a component of the export apparatus (Collier, D.N., Bankaitis, V.A., Weiss, J.B., and Bassford, P.J. (1988) Cell 53, 273-283). Consistent with this observation, we find that the interference elicited by an export-defective LamB protein is a titratable response resulting from the limitation of a single ligand. We have mapped the interfering region in LamB to between amino acids 320 and 380 of the mature protein. Expression of this sequence in the form of a LacZ-LamB-LacZ fusion protein elicits the export interference phenotype. Deletion of the sequence from an export-defective LamB protein eliminates the ability of this protein to interfere with the export of other secreted proteins. Together, these findings show that this sequence is both necessary and sufficient to cause export interference. Surprisingly, deletion of this sequence from an otherwise wild-type LamB protein does not cause the mutant LamB product to exhibit any obvious export defect. Based on our results, we propose that SecB interacts with both amino acids 320-380 of mature LamB and the LamB signal sequence during initiation of the export process.     

Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein

We previously described a dominant negative secY ^-d 1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY. Syd is the product of a multicopy suppressor of the secY ^-d 1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein. In contrast, overproduction of Syd is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240→Asp). The secY249 mutation (Ala249→Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation. The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction. The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein. Although the secY245 mutant retained some sensitivity?to Syd overproduction, the secY241 mutant was completely Syd-resistant. Furthermore, the secY241 mutation seemed to represent a “hyper reversion” with respect to the SecY-SecE interaction. Protein export in this mutant was no longer sensitive to SecY^-d1. When the secY ^-d 1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY ^-d 1–241) showed an increased ability to interfere with protein export. On the basis of our model for SecY^-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction. These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex.