Role of the non-essential region encompassing the N-terminal two transmembrane stretches of Escherichia coli SecE

SecE is an essential component of the protein translocation machinery of Escherichia coli and has three transmembrane stretches. An N-terminal region (SecE-N) encompassing the first two transmembrane stretches is dispensable for protein translocation but a SecE derivative (SecE-C) lacking this region is very unstable. We show here that FtsH, the AAA (ATPases associated with diverse cellular activities) family protease, causes the instability of SecE-C. SecE-C became stable when SecE-N was co-expressed. Deletion of the N-terminal region of SecE also rendered the SecE-SecY-SecG complex unstable. In spite of these alterations, the N-terminal region of SecE had little stimulatory effect on protein translocation in vivo or in vitro.     

The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation

We have previously reconstituted the soluble phase of precursor protein translocation in vitro using purified proteins (the precursor proOmpA, the chaperone SecB, and the ATPase SecA) in addition to isolated inner membrane vesicles. We now report the isolation of the SecY/E protein, the integral membrane protein component of the E. coli preprotein translocase. The SecY/E protein, reconstituted into proteoliposomes, acts together with SecA protein to support translocation of proOmpA, the precursor form of outer membrane protein A. This translocation requires ATP and is strongly stimulated by the protonmotive force. The initial rates and the extents of translocation into either native membrane vesicles or proteoliposomes with pure SecY/E are comparable. The SecY/E protein consists of SecY, SecE, and an additional polypeptide. Antiserum against SecY immunoprecipitates all three components of the SecY/E protein.     

Regulation of a membrane component required for protein secretion in Escherichia coli

We have previously described a gene, secA, which may code for a component of the secretion machinery of E. coli. Temperature-sensitive mutations in this gene lead to the cytoplasmic accumulation of precursors to a number of secreted proteins. In this paper, we describe the use of antibody to the SecA protein to characterize the cellular location and regulation of the protein. The antibody was elicited in response to a SecA-LacZ hybrid protein, produced by a strain carrying a secA-lacZ gene fusion. The secA gene product is a 92 kd polypeptide that is present in small amounts in the cell and that fractionates as a peripheral cytoplasmic membrane protein. The synthesis of the SecA protein is greatly derepressed (at least tenfold) when secretion in E. coli is blocked either in a secAts mutant or in the presence of a MalE-LacZ hybrid protein. We suggest that components of the secretion machinery of E. coli, such as the SecA protein, may be regulated in response to the secretion needs of the cell. When suppression of a secAam mutant is eliminated, leading to the absence of SecA protein, the synthesis of maltose-binding protein is greatly reduced. These results support a mechanism in which secretion and translation are coupled.     

The MotA protein of E. coli is a proton-conducting component of the flagellar motor

A number of mutants of motA, a gene necessary for flagellar rotation in E. coli, were isolated and characterized. Many mutations were dominant, owing to competition between functional and nonfunctional MotA for a limited number of sites on the flagellar motor. A new class of mutant was discovered in which flagellar torque is normal at low speeds but reduced at high speeds. Hydrogen isotope effects on these mutants indicate that MotA catalyzes proton transfer. We confirmed an earlier observation that overproduction of MotA leads to accumulation of the protein in the cytoplasmic membrane and to significant decreases in growth rate. When nonfunctional mutant variants of MotA were overproduced instead, they accumulated in the cytoplasmic membrane, but growth was not impaired. These results also suggest that MotA conducts protons. This was confirmed by measuring the proton permeabilities of vesicles containing wild-type or mutant MotA proteins.     

SecY is an indispensable component of the protein secretory machinery of Escherichia coli

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.     

SecA protein is required for secretory protein translocation into E. coli membrane vesicles

The soluble and membrane components of an E. coli in vitro protein translocation system prepared from a secA amber mutant, secA13[Am], contain reduced levels of SecA and are markedly defective in both the cotranslational and posttranslational translocation of OmpA and alkaline phosphatase into membrane vesicles. Moreover, the removal of SecA from soluble components prepared from a wild-type strain by passage through an anti-SecA antibody column similarly abolishes protein translocation. Translocation activity is completely restored by addition of submicrogram amounts of purified SecA protein, implying that the observed defects are solely related to loss of SecA function. Interestingly, the translocation defect can be overcome by reconstitution of SecA into SecA-depleted membranes, suggesting that SecA is an essential, membrane-associated translocation factor.     

Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion

Localization of RNA replication to intracellular membranes is a universal feature of positive-strand RNA viruses. Replication complexes of flock house virus (FHV), the best-studied alphanodavirus, are located on outer mitochondrial membranes in infected Drosophila melanogaster cells and are associated with the formation of membrane-bound spherules, similar to structures found for many other positive-strand RNA viruses. To further study FHV replication complex formation, we investigated the subcellular localization, membrane association, and membrane topology of protein A, the FHV RNA-dependent RNA polymerase, in the yeast Saccharomyces cerevisiae, a host able to support full FHV RNA replication and virion formation. Confocal immunofluorescence revealed that protein A localized to mitochondria in yeast, as in Drosophila cells, and that this mitochondrial localization was independent of viral RNA synthesis. Nycodenz gradient flotation and dissociation assays showed that protein A behaved as an integral membrane protein, a finding consistent with a predicted N-proximal transmembrane domain. Protease digestion and selective permeabilization after differential epitope tagging demonstrated that protein A was inserted into the outer mitochondrial membrane with the N terminus in the inner membrane space or matrix and that the C terminus was exposed to the cytoplasm. Flotation and immunofluorescence studies with deletion mutants indicated that the N-proximal region of protein A was important for both membrane association and mitochondrial localization. Gain-of-function studies with green fluorescent protein fusions demonstrated that the N-terminal 46 amino acids of protein A were sufficient for mitochondrial localization and membrane insertion. We conclude that protein A targets and anchors FHV RNA replication complexes to outer mitochondrial membranes, in part through an N-proximal mitochondrial localization signal and transmembrane domain.     

The unique transmembrane hairpin of flavivirus fusion protein E is essential for membrane fusion

The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion.     

Identification of the outer membrane protein of E.coli that produces transmembrane channels in reconstituted vesicle membranes

Outer membrane of $\text{Escherichia}$$\text{coli}$ allows a rapid diffusion of saccharides of molecular weights less than 550. This permeability property could be restored in vesicle membranes reconstituted from isolated phospholipids, lipopolysaccharide, and an outer membrane protein. The active protein aggregates were isolated from the insoluble material left after solubilization of cell envelope of $\text{Escherichia}$$\text{coli}$ B with sodium dodecyl sulfate at 35°. Analysis by acrylamide gel electrophoresis, isoelectric focusing and amino terminal amino acid determination revealed that only a single species of protein, with a molecular weight of 36,500 forms the oligoprotein aggregates which produces diffusion channels.     

Role of the mature protein sequence of maltose-binding protein in its secretion across the E. coli cytoplasmic membrane

Secretion of amber fragments of an E. coli periplasmic protein, the maltose-binding protein, was studied to determine if the mature portion of the protein is required for its export across the cytoplasmic membrane. A fragment lacking 25–35 amino acid residues at the C terminus is secreted at normal levels, suggesting that this sequence is not required for secretion. This is in contrast to the results obtained with the periplasmic protein β-lactamase. In studying another fragment of one-third the molecular weight of the intact protein, we found that the majority of the fragment is not recovered from the periplasmic fraction. However, a small amount of secretion of this polypeptide was observed. This fragment is synthesized as a larger molecular weight form when cells are induced for the synthesis of a maltose-binding protein-β-galactosidase hybrid protein, which was previously shown to block the proper localization and processing of envelope proteins. This result is consistent with the idea that the larger form is a precursor with an unprocessed signal sequence, whereas in the absence of the hybrid protein the fragment is a processed mature form. Thus secretion of the smaller fragment may be occurring up to the point where the signal sequence is removed. That this fragment has passed through the cytoplasmic membrane is further supported by its accessibility to externally added trypsin. We suggest that the fragment may be secreted to the periplasm, but cannot assume a water-soluble conformation; the majority of the polypeptide may be associated with the external surface of the cytoplasmic membrane. Thus the mature sequence of maltose-binding protein, at least its C-terminal two thirds, may not be required for its export across the cytoplasmic membrane.     

The inner membrane protein, YhiM, is necessary for Escherichia coli (E. coli) survival in acidic conditions

Escherichia coli must be able to survive extreme acidic conditions. We were interested in determining the role of the inner membrane protein YhiM in survival in acidic conditions. Previous data demonstrated that the yhiM gene was upregulated in acidic conditions (Tucker et al. in J Bacteriol. 184:6551-6558, 2002). We therefore tested tn10 insertions into the yhiM gene for their ability to survive at low pH (pH 2.5). We show that YhiM was required for survival at pH 2.5. We also tested the YhiM dependence of the different acid resistance pathways. YhiM was required for the RpoS, glutamine and lysine-dependent acid resistance pathways. In contrast, YhiM was not required for the arginine-dependent acid resistance pathway.     

The N-terminal region of the Escherichia coli WecA (Rfe) protein, containing three predicted transmembrane helices, is required for function but not for membrane insertion

The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.     

The genetics of protein secretion in E. coli

Genetic studies have identified six genes whose products comprise the general protein secretion machinery of Escherichia coli. Insights from mutant analysis and the biochemical properties of the purified components allows the secretion pathway to be described in some detail. The picture emerging provides a useful paradigm for similar pathways in other organisms.     

Topology of IEP110, a component of the chloroplastic protein import machinery present in the inner envelope membrane.

Proteins from both the inner and outer envelope membranes are engaged in the recognition and translocation of precursor proteins into chloroplasts. A 110 kDa protein of the chloroplastic inner envelope membrane was identified as a component of the protein import apparatus by two methods. First, this protein was part of a 600 kDa complex generated by cross-linking of precursors trapped in the translocation process. Second, solubilization with detergents of chloroplasts containing trapped precursors resulted in the identification of a complex containing both radiolabeled precursor and IEP110. Trypsin treatment of intact purified chloroplasts was used to study the topology of IEP110. The protease treatment left the inner membrane intact while simultaneously degrading domains of inner envelope proteins exposed to the intermembrane space. About 90 kDa of IEP110 was proteolitically removed, indicating that large portions protrude into the intermembrane space. Hydropathy analysis of the protein sequence deduced from the isolated cDNA clone in addition to Western blot analysis using an antiserum of IEP110 specific to the N-terminal 20 kDa, suggests that the N-terminus serves to anchor the protein in the membrane. We speculate that IEP110 could be involved in the formation of translocation contact sites due to its specific topology.     

The outer membrane component, YscC, of the Yop secretion machinery of Yersinia enterocolitica forms a ring-shaped multimeric complex

The YscC protein of Yersinia enterocolitica is essential for the secretion of anti-host factors, called Yops, into the extracellular environment. It belongs to a family of outer membrane proteins, collectively designated secretins, that participate in a variety of transport processes. YscC has been shown to exist as a stable oligomeric complex in the outer membrane. The production of the YscC complex is regulated by temperature and is reduced in strains carrying mutations in the yscN-U operon or in the virG gene. The VirG lipoprotein was shown to be required for efficient targeting of the complex to the outer membrane. Electron microscopy revealed that purified YscC complexes form ring-shaped structures of ≈20 nm with an apparent central pore. Because of the architecture of the multimer, YscC appears to represent a novel type of channel-forming proteins in the bacterial outer membrane.     

Escherichia coli outer membrane protein K is a porin.

Protein K is an outer membrane protein found in pathogenic encapsulated strains of Escherichia coli. We present evidence here that protein K is structurally and functionally related to the E. coli K-12 porin proteins (OmpF, OmpC, and PhoE). Protein K was found to cross-react with antibody to OmpF protein and to share 8 out of 17 peptides in common with the OmpF protein. Strains that are OmpC porin- and OmpF porin- and contain protein K as their major outer membrane protein have increased rates of uptake of nutrients and a faster growth rate relative to the parental porin- strain. The protein K-containing strains are at least 1,000-fold more sensitive to colicins E2 and E3 than is the porin -deficient strain. These data suggest that protein K is a functional porin in E. coli. The porin function of protein K was also demonstrated in vitro, using black lipid membranes. Protein K increased the conductance in these membranes in discrete, uniform steps characteristic of channels with a size of about 2 nS.     

The signal Peptide of a vacuolar protein is necessary and sufficient for the efficient secretion of a cytosolic protein

A cytosolic pea (Pisum sativum) seed albumin (ALB) and a chimeric protein (PHALB) consisting of the signal peptide and first three amino acids of phytohemagglutinin (PHA) and the amino acid sequence of ALB were expressed in parallel suspension cultures of tobacco (Nicotiana tabacum) cells and their intracellular fates examined. PHALB was efficiently secreted by the cells whereas ALB remained intracellular. These experiments show that the information contained in the signal peptide of a vacuolar protein is both necessary and sufficient for efficient secretion, and define secretion as a default or bulk-flow pathway. Entry into the secretory pathway was accompanied by glycosylation and the efficient conversion of the high mannose glycans into complex glycans indicating that transported glycoproteins do not need specific recognition domains for the modifying enzymes in the Golgi. Tunicamycin depressed the accumulation of the unglycosylated polypeptide in the culture medium much less than the accumulation of other glycoproteins. We interpret this as evidence that glycans on proteins that are not normally glycosylated do not have the same function of stabilizing and protecting the polypeptide as on natural glycoproteins.     

Glycosylation of a membrane protein is restricted to the growing polypeptide chain but is not necessary for insertion as a transmembrane protein.

The membrane glycoprotein of vesicular stomatitis virus (VSV), synthesized in vitro in the presence of pancreatic microsomes, is glycosylated in two distinct steps while its polypeptide chain is nascent (Rothman and Lodish, 1977). We show here that unglycosylated glycoprotein, which accumulates in vivo following treatment of cells with tunicamycin and in vitro as a result of translation in the presence of detergent-treated microsomal membranes, is inserted normally as a transmembrane protein. This means that glycosylation, while normally occurring concurrently with insertion, is not required for insertion. Our experiments also show that the two steps in glycosylation correspond to the sequential transfer of preformed “core” oligosaccharides of typical structure to two Asn residues in the growing chain. The accumulation of unglycosylated glycoprotein in vitro is due to the fact that the completed transmembrane polypeptide cannot be glycosylated. The detergent treatment of microsomes impairs their rate of glycosylation so that chains are frequently completed before they can be glycosylated. This provides a simple explanation for certain types of heterogeneity often found in glycoproteins. We believe that the detergent treatment procedure results in the solubilization of the microsomal membrane followed by reconstitution. This is a prerequisite for the eventual purification of the membrane proteins and lipids involved in insertion and glycosylation of this model membrane protein.     

The C terminus of penicillin-binding protein 5 is essential for localisation to the E. coli inner membrane.

Penicillin-binding protein 5 (PBP5) has been previously identified as a component of the inner membrane of Escherichia coli and we present here further evidence that PBP5 is tightly bound to the membrane. To investigate the regions of PBP5 involved in membrane binding we have constructed a series of C-terminal deletions and shown that the removal of as few as 10 amino acids results in the release of the truncated protein into the periplasm. The C terminus, therefore, appears to be important for interaction with the membrane; however, inspection of the amino acid sequence does not reveal extended runs of hydrophobicity typical of a membrane anchor. Thus we conclude that PBP5 is anchored to the inner membrane by a mechanism not previously described.