sn-glycerol-3-phosphate acyltransferase tubule formation is dependent upon heat shock proteins (htpR)

Overexpression of the Escherichia coli sn-glycerol-3-phosphate (glycerol-P) acyltransferase, an integral membrane protein, causes formation of ordered arrays of the enzyme in vitro. The formation of these tubular structures did not occur in an E. coli strain bearing a mutation in the htpR gene, the regulatory gene for the heat shock response. The htpR165 mutation was shown by genetic analysis to be the lesion responsible for blockage of tubule formation. Similar amounts of glycerol-P acyltransferase were produced in isogenic htpR+ and htpR165 strains, ruling out an effect of htpR165 on expression of glycerol-P acyltransferase. Further, phospholipid metabolism was not altered in either strain after induction of glycerol-P acyltransferase synthesis. Increased glycerol-P acyltransferase synthesis caused a partial induction of the heat shock response which was dependent upon a wild type htpR gene. The heat shock proteins induced were identified as the groEL and dnaK gene products on two-dimensional gels. These two proteins have been implicated in the assembly of bacteriophage coats. These heat shock proteins appear essential for tubule formation.     

Thermo-regulated expression of the htpR gene under the control of the PR-promotor of bacteriophage lambda induces supersynthesis of heat shock proteins

The mechanisms of induction of heat shock protein synthesis in E. coli have been studied. For this purpose plasmids in which htpR gene expression is controlled by the PR-promoter of bacteriophage lambda and by the Trp-promoter have been constructed. An effective induction of heat shock proteins requires both an increased content of htpR protein and additional cofactors formed in the cell under heat shock conditions.     

The mature portion of Escherichia coli maltose-binding protein (MBP) determines the dependence of MBP on SecB for export.

The product of the secB gene is required for export of a subset of secreted proteins to the outer membrane and periplasm of Escherichia coli. Precursor maltose-binding protein (MBP) accumulates in the cytoplasm of secB-carrying mutants, but export of alkaline phosphatase is only minimally affected by secB mutations. When export of MBP-alkaline phosphatase hybrid proteins was analyzed in wild-type and secB-carrying mutant strains, the first third of mature MBP was sufficient to render export of the hybrid proteins dependent on SecB. Substitution of a signal sequence from a SecB-independent protein had no effect on SecB-dependent export. These findings show that the first third of mature MBP is capable of conferring export incompetence on an otherwise competent protein.     

Protein localization in Escherichia coli K-12: an analysis of ompC-lacZ gene fusions

Abstract Two conditionally expressed lacZU131 gene fusions were constructed in vivo to the ompC gene which encodes a major outer membrane protein in Escherichia coli . The resulting hybrid molecules contained approximately 25% and 50% of the mature OmpC protein fused to the LacZ. Export analysis showed that under nonoverproducing conditions essentially all synthesized OmpC-LacZ hybrid protein was effectively processed in vivo unless the signal peptide cleavage was inhibited by ethanol addition. Also, the hybrid proteins were highly accessible to solid phase iodination of whole cells under conditions where cytoplasmic proteins remained unlabelled. Thus, hybrids containing large portions of the OmpC protein were clearly recognized by the cellular export machinery, and probably all synthesized hybrid protein was partially translocated through the cytoplasmic membrane.     

Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.     

Anticipating endoplasmic reticulum stress. A novel early response before pathogenesis-related gene induction

When it is attacked by a pathogen, a plant produces a range of defense-related proteins. Many of these are synthesized by the rough endoplasmic reticulum (RER) to be secreted from the cell or deposited in vacuoles. Genes encoding endoplasmic reticulum (ER)-resident chaperones, such as the lumenal binding protein (BiP), are also induced under these conditions. Here, we show that BiP induction occurs systemically throughout the plant. Furthermore, this induction occurs rapidly and precedes expression of genes encoding pathogenesis-related (PR) proteins. The underlying signal transduction pathway was shown to be independent of the signaling molecule salicylic acid and the unfolded protein response pathway. In addition, BiP induction was independent of PR gene induction. Overproduction of BiP alone was not sufficient to cause induction of PR gene expression; however, limiting the amount of BiP in the ER lumen via superimposed ER stress inhibited the induction of PR gene expression. We propose that the induction of BiP expression during plant-pathogen interactions is required as an early response to support PR protein synthesis on the RER and that a novel signal transduction pathway exists to trigger this rapid response.     

Green fluorescent protein functions as a reporter for protein localization in Escherichia coli

The use of green fluorescent protein (GFP) as a reporter for protein localization in Escherichia coli was explored by creating gene fusions between malE (encoding maltose-binding protein [MBP]) and a variant of gfp optimized for fluorescence in bacteria (GFPuv). These constructs encode hybrid proteins composed of GFP fused to the carboxy-terminal end of MBP. Fluorescence was not detected when the hybrid protein was synthesized with the MBP signal sequence. In contrast, when the MBP signal sequence was deleted, fluorescence was observed. Cell fractionation studies showed that the fluorescent MBP-GFP hybrid protein was localized in the cytoplasm, whereas the nonfluorescent version was localized to the periplasmic space. Smaller MBP-GFP hybrid proteins, however, exhibited abnormal fractionation. Expression of the gene fusions in different sec mutants, as well as signal sequence processing assays, confirmed that the periplasmically localized hybrid proteins were exported by the sec-dependent pathway. The distinction between fluorescent and nonfluorescent colonies was exploited as a scorable phenotype to isolate malE signal sequence mutations. While expression of hybrid proteins comprised of full-length MBP did not result in overproduction lethality characteristic of some exported beta-galactosidase hybrid proteins, synthesis of shorter, exported hybrid proteins was toxic to the cells. Purification of MBP-GFP hybrid protein from the different cellular compartments indicated that GFP is improperly folded when localized outside of the cytoplasm. These results suggest that GFP could serve as a useful reporter for genetic analysis of bacterial protein export and of protein folding.     

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.     

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.     

Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli.

The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export. We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation. Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2. The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex. The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215. The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized. This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein. These results suggest that IF2 or the translation initiation step can modulate protein export reactions. The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E. coli cells.     

Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway

Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.     

Molecular cloning and expression of a gene that controls the high-temperature regulon of Escherichia coli.

The high-temperature production (HTP) regulon of Escherichia coli consists of a set of operons that are induced coordinately by a shift to a high temperature under the control of a single chromosomal gene called htpR or hin. To identify more components of this regulon, the rates of synthesis of many polypeptides resolved on two-dimensional polyacrylamide gels were measured in various strains by pulse-labeling after a temperature shift-up. A total of 13 polypeptides were found to be heat inducible only in cells bearing a normal htpR gene on the chromosome or on a plasmid; on this basis these polypeptides were designated products of the HTP regulon. Several hybrid plasmids that contain segments of the E. coli chromosome in the 75-min region were found to carry the htpR gene. A restriction map of this region was constructed, and selected fragments were subcloned and tested for the ability to complement an htpR mutant. The polypeptides encoded by these fragments were detected by permitting expression in maxicells, minicells, and chloramphenicol-treated cells. Complementation was accompanied by production of a polypeptide having a molecular weight of approximately 33,000. This polypeptide, designated F33.4, was markedly reduced in amount in an htpR mutant expected to contain very little htpR gene product. Polypeptide F33.4 is postulated to be the product of htpR and to be an effector that controls heat induction of the HTP regulon.     

Overlapping functions of components of a bacterial Sec-independent protein export pathway.

We describe the identification of two Escherichia coli genes required for the export of cofactor-containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor-containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.     

Targeting of signal sequenceless proteins for export in Escherichia coli with altered protein translocase.

Most extracytoplasmic proteins are synthesized with an N-terminal signal sequence that targets them to the export apparatus. Escherichia coli prlA mutants (altered in the secY gene) are able to export cell envelope proteins lacking any signal sequence. In order to understand how such proteins are targeted for export, we isolated mutations in a signal sequenceless version of alkaline phosphatase that block its export in a prlA mutant. The mutations introduce basic amino acyl residues near the N-terminus of alkaline phosphatase. These changes do not disrupt an N-terminal export signal in this protein since the first 25 amino acids can be removed without affecting its export competence. These findings suggest that signal sequenceless alkaline phosphatase does not contain a discrete domain that targets it for export and may be targeted simply because it remains unfolded in the cytoplasm. We propose that basic amino acids near the N-terminus of a signal sequenceless protein affect its insertion into the translocation apparatus after it has been targeted for export. These findings allow the formulation of a model for the entry of proteins into the membrane-embedded export machinery.     

Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli.

The 42-1 lamB-lacZ gene fusion confers a conditionally lethal, export-dependent phenotype known as maltose sensitivity. A maltose-resistant mutant showing decreased beta-galactosidase activity of the hybrid protein, designated prlF1 (protein localization), was unlinked to the lamB-lacZ fusion. This mutation mapped at 70 min on the Escherichia coli linkage map and conferred maltose resistance, a 30-fold reduction in beta-galactosidase activity, and a 30% decrease in cellular growth rate at 30 degrees C that was independent of the presence of a gene fusion. prlF1 also decreased the beta-galactosidase activity and relieved the maltose sensitivity conferred by fusions of lacZ to the gene specifying the periplasmic maltose-binding protein, malE. The decrease in beta-galactosidase activity, however, was specific for exported hybrid proteins. When export of the hybrid protein was blocked by a signal sequence mutation, prlF1 decreased the beta-galactosidase activity only 2.5-fold. Similarly, prlF1 did not affect the beta-galactosidase activity of fusions of lacZ to a gene specifying a nonexported protein, malK.     

SecB-independent export of Escherichia coli ribose-binding protein (RBP): some comparisons with export of maltose-binding protein (MBP) and studies with RBP-MBP hybrid proteins.

The efficient export of the Escherichia coli maltose-binding protein (MBP) is known to be SecB dependent, whereas ribose-binding protein (RBP) export is SecB independent. When the MBP and RBP signal peptides were exchanged precisely at the signal peptidase processing sites, the resultant RBP-MBP and MBP-RBP hybrid proteins both were efficiently exported in SecB+ cells. However, only MBP-RBP was efficiently exported in SecB- cells; RBP-MBP exhibited a significant export defect, a finding that was consistent with previous proposals that SecB specifically interacts with the mature moiety of precursor MBP to promote export. The relatively slow, totally posttranslational export mode exhibited by certain mutant RBP and MBP-RBP species in SecB+ cells was not affected by the loss of SecB. In contrast, MBP and RBP-MBP species with similarly altered signal peptides were totally export defective in SecB- cells. Both export-defective MBP and RBP-MBP interfered with SecB-mediated protein export by depleting cells of functional SecB. In contrast, neither export-defective RBP nor MBP-RBP elicited such an interference effect. These and other data indicated that SecB is unable to interact with precursor RBP or that any interaction between these two proteins is considerably weaker than that of SecB with precursor MBP. In addition, no correlation could be established between a SecB requirement for export and PrlA-mediated suppression of signal peptide export defects. Finally, previous studies have established that wild-type MBP export can be accomplished cotranslationally, whereas wild-type RBP export is strictly a posttranslational process. In this study, cotranslational export was not detected for either MBP-RBP or RBP-MBP. This indicates that the export mode exhibited by a given precursor protein (cotranslational versus posttranslational) is determined by properties of both the signal peptide and the mature moiety.     

Translational control of exported proteins that results from OmpC porin overexpression.

The regulation of synthesis and export of outer membrane proteins of Escherichia coli was examined by overexpressing ompC in multicopy either from its own promoter or from an inducible promoter in an expression vector. Overexpression of OmpC protein resulted in a nearly complete inhibition of synthesis of the OmpA and LamB outer membrane proteins but had no effect on synthesis of the periplasmic maltose-binding protein. Immunoprecipitation of labeled proteins showed no evidence of accumulation of uncleaved precursor forms of OmpA or maltose-binding protein following induction of OmpC overexpression. The inhibition of OmpA and LamB was tightly coupled to OmpC overexpression and occurred very rapidly, reaching a high level within 2 min after induction. OmpC overexpression did not cause a significant decrease in expression of a LamB-LacZ hybrid protein produced from a lamB-lacZ fusion in which the fusion joint was at the second amino acid of the LamB signal sequence. There was no significant decrease in rate of synthesis of ompA mRNA as measured by filter hybridization of pulse-labeled RNA. These results indicate that the inhibition is at the level of translation. We propose that cells are able to monitor expression of exported proteins by sensing occupancy of some limiting component in the export machinery and use this to regulate translation of these proteins.