The ultimate localization of an outer membrane protein of Escherichia coli K-12 is not determined by the signal sequence

To study the role of the signal sequences in the biogenesis of outer membrane proteins, we have constructed two hybrid genes: a phoE-ompF hybrid gene, which encodes the signal sequence of outer membrane PhoE protein and the structural sequence of outer membrane OmpF protein, and a bla-phoE hybrid gene which encodes the signal sequence as well as 158 amino acids of the structural sequence of the periplasmic enzyme beta-lactamase and the complete structural sequence of PhoE protein. The products of these genes are normally transported to and assembled into the outer membrane These results show: (i) that signal sequences of exported proteins are export signals which function independently of the structural sequence, and (ii) that the information which determines the ultimate location of an outer membrane protein is located in the structural sequence of this protein, and not in the signal sequence.     

On the role of the mature part of an Escherichia coli outer membrane protein (OmpA) in translocation across the plasma membrane

The 325-residue OmpA protein, which is synthesized as a precursor with a 21-residue signal sequence, is a polypeptide of the outer membrane of Escherichia coli K-12. The signal peptide is able to direct translocation across the plasma membrane of virtually any fragment of this protein. It had, therefore, been concluded that information required for this translocation does not exist within the mature part of the protein. This view has been criticized and it was suggested that our data showed that both the signal sequence and residues within the first 44 amino acid residues of the mature protein contributed to an optimal translocation mechanism. It is shown that, at least as far as is detectable, this is not so. The apparent rates of processing of various pro-OmpA constructs were measured. It was found that these rates did not depend on the presence of amino acid residues 4 through 45 but on the size of the polypeptides; the processing rate decreased with decreasing size. A possible explanation for this phenomenon is offered. While the results do not exclude the possibility that a defined area of the mature protein is involved in optimizing translocation, there is so far no evidence for it.     

An inner membrane protein N-terminal signal sequence is able to promote efficient localisation of an outer membrane protein in Escherichia coli

To test the importance of N-terminal pre-sequences in translocation of different classes of membrane proteins, we exchanged the normal signal sequence of an Escherichia coli outer membrane protein, OmpF, for the pre-sequence of the inner membrane protein, DacA. The DacA-OmpF hybrid was efficiently assembled into the outer membrane in a functionally active form. Thus the pre-sequence of DacA, despite its relatively low hydrophobicity compared with that of OmpF, contains all the essential information necessary to initiate the translocation of OmpF to the outer membrane. Since processing of DacA was also shown to be dependent upon SecA we conclude that the initiation of translocation of this inner membrane polypeptide across the envelope occurs by the same mechanism as outer membrane and periplasmic proteins. The N-terminal 11 amino acids of mature OmpF, which in the hybrid are replaced by the N-terminal nine amino acids of DacA, carry no essential assembly signals since the hybrid protein is apparently assembled with equal efficiency to OmpF.     

Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.     

Energy requirements for protein translocation across the Escherichia coli inner membrane

Both ATP and an electrochemical potential play roles in translocating proteins across the inner membrane of Escherichia coli. Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy-requiring steps of the reaction from purified components. A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier.     

Molecular chaperones and protein translocation across the Escherichia coli inner membrane

Proteins that are able to translocate across biological membranes assume a loosely folded structure. In this review it is suggested that the loosely folded structure, referred to here as the 'pre-folded conformation', is a particular structure that interacts favourably with components of the export apparatus. Two soluble factors, SecB and GroEL, have been implicated in maintenance of the pre-folded conformation and have been termed 'molecular chaperones'. Results suggest that SecB may be a chaperone that is specialized for binding to exported protein precursors, while GroEL may be a general folding modulator that binds to many intracellular proteins.     

The signal sequence suffices to direct export of outer membrane protein OmpA of Escherichia coli K-12.

We studied whether information required for export is present within the mature form of the Escherichia coli 325-residue outer membrane protein OmpA. We had previously analyzed overlapping internal deletions in the ompA gene, and the results allowed us to conclude that if such information exists it must be present repeatedly within the membrane part of the protein encompassing amino acid residues 1 to 177 (R. Freudl, H. Schwarz, M. Klose, N. R. Movva, and U. Henning, EMBO J. 4:3593-3598, 1985). A deletion which removed the codons for amino acid residues 1 to 229 of the OmpA protein was constructed. In this construct the signal sequence was fused to the periplasmic part of the protein. The resulting protein, designated Pro-OmpA delta 1-229, was processed, and the mature 95-residue protein accumulated in the periplasm. Hence, information required for export does not exist within the OmpA protein.     

Region of heat-stable enterotoxin II of Escherichia coli involved in translocation across the outer membrane

Heat-stable enterotoxin II of Escherichia coli (STII) is synthesized as a precursor form consisting of pre- and mature regions. The pre-region is cleaved off from the mature region during translocation across the inner membrane, and the mature region emerges in the periplasm. The mature region, composed of 48 amino acid residues, is processed in the periplasm by DsbA to form an intramolecular disulfide bond between Cys-10 and Cys-48 and between Cys-21 and Cys-36. STII formed with these disulfide bonds is efficiently secreted out of the cell through the secretory system, including TolC. However, it remains unknown which regions of STII are involved in interaction with TolC. In this study, we mutated the STII gene and examined the secretion of these STIIs into the culture supernatant. A deletion of the part covering from amino acid residue 37 to the carboxy terminal end did not markedly reduce the efficiency of secretion of STII into the culture supernatant. On the other hand, the efficiency of secretion of the peptide covering from the amino terminal end to position 18 to the culture supernatant was significantly low. These observations indicated that the central region of STII from amino acid residue 19 to that at position 36 is involved in the secretion of STII into the milieu. The experiment using a dsbA-deficient strain of E. coli showed that the disulfide bond between Cys-21 and Cys-36 by DsbA is necessary for STII to adapt to the structure that can cross the outer membrane.     

An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtllis

The translocation of secretory proteins derived from a Gram-positive (Staphylococcus hyicus prolipase) or a Gram-negative (Escherichia coli pre-OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. in both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre-prolipase signal peptide (38 amino acids in length) or the pre-OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre-OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram-positive and Gram-negative bacteria.     

A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli.

A novel factor, which is a membrane component of the protein translocation machinery of Escherichia coli, was discovered. This factor was found in the trichloracetic acid-soluble fraction of solubilized cytoplasmic membrane. The factor was purified to homogeneity by ion exchange column chromatographies and found to be a hydrophobic protein with a molecular mass of approximately 12 kDa. The factor caused > 20-fold stimulation of the protein translocation when it was reconstituted into proteoliposomes together with SecE and SecY. SecE, SecY, SecA and ATP were essential for the factor-dependent stimulation of the activity. The factor stimulated the translocation of all three precursor proteins examined, including authentic proOmpA. Stimulation of the translocation of proOmpF-Lpp, a model presecretory protein, was especially remarkable, since no translocation was observed unless proteoliposomes were reconstituted with the factor. Partial amino acid sequence of the purified factor was determined. An antibody raised against a synthetic peptide of this sequence inhibited the protein translocation into everted membrane vesicles, indicating that the factor is playing an important role in protein translocation into membrane vesicles. The partial amino acid sequence was found to coincide with that deduced from the reported DNA sequence of the upstream region of the leuU gene. Cloning and sequencing of the upstream region revealed the presence of a new open reading frame, which encodes a hydrophobic protein of 11.4 kDa. We propose that the factor is a general component of the protein translocation machinery of E. coli.     

Characterization of the interfacial behavior and structure of the signal sequence of Escherichia coli outer membrane pore protein PhoE

The behavior of the chemically synthesized PhoE signal peptide and signal peptide fragments on hydrophilic-hydrophobic interfaces was studied with circular dichroism and monolayer techniques. The experimental results were compared with computer-calculated predictions of peptide structure, orientation, and molecular area. The complete signal sequence was found to aggregate in a beta-sheet structure when introduced in an aqueous environment; on the other hand, in sodium dodecyl sulfate micelles approximately 75% alpha-helical structure was observed. Assuming this to reflect the actual structure in a peptide monolayer and taking into account the orientations predicted for the fragments, the measured molecular areas suggest a looped orientation of the signal sequence with both N and C terminus in the water phase.     

SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane.

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.     

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.     

An artificial hydrophobic sequence functions as either an anchor or a signal sequence at only one of two positions within the Escherichia coli outer membrane protein OmpA

The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids. A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively. In the first case, the lipophilic sequence anchored the protein in the plasma membrane. In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor. By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence. Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4. Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA. Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly.     

PrlC, a suppressor of signal sequence mutations in Escherichia coli, can direct the insertion of the signal sequence into the membrane

The prlC gene product of Escherichia coli can be altered by mutation so that it restores export of proteins with defective signal sequences. The strongest suppressor, prlC8, restores processing of a mutant signal sequence to a rate indistinguishable from the wild-type. Data obtained by changing gene dosage of the dominant suppressor and its specificity for different signal sequence mutations suggest that PrlC8 interacts directly with the hydrophobic core of the signal sequence. Despite the fact that signal sequence processing appears to be mediated by leader peptidase, the processed mature protein is not translocated efficiently from the cytoplasm. Results obtained with various double mutants indicate that PrlC8-mediated processing of mutant signal sequences does not require components of the cellular export machinery such as SecA, SecB or PrlA (SecY) and that the block in translocation from the cytoplasm occurs because PrlA (SecY) fails to recognize the defective signal sequence. We suggest that PrlC8 directs insertion of the mutant signal sequence into the membrane bilayer to an extent that processing by leader peptidase can occur. This reaction is novel in that it has not been observed previously in vivo.     

Three-dimensional structure of Wza, the protein required for translocation of group 1 capsular polysaccharide across the outer membrane of Escherichia coli

Wza is a highly conserved multimeric outer membrane protein complex required for the surface expression of the serotype K30 group 1 capsular polysaccharide in Escherichia coli. Here we present the first three-dimensional structure of this type of polysaccharide exporter at a 15.5-A resolution obtained using single particle averaging on a dataset of cryo-negatively stained protein. Previous structural studies on purified Wza have revealed a homo-oligomeric ring structure that is most probably composed of eight subunits. Symmetry analysis of the three-dimensional structure combined with biochemical two- and three-dimensional crystallographic data strongly suggest that Wza is an octameric complex with a C4 quasi-rotational symmetry and is organized as a tetramer of dimeric subunits. Wza is best described as a stack of two 4-A high rings with differing diameters providing a mushroom-like aspect from the side. The larger ring has a distinctive square shape with a diameter of 115 A, whereas the smaller is almost circular with a diameter of 90 A. In the center of the complex and enclosed by the four symmetrical arms is a small elliptical cagelike cavity of approximately 40 A in diameter. The central cavity is effectively sealed at the top and bottom of the complex but has small inter-arm holes when viewed from the side. We discuss the structure of this complex and implications in the surface translocation of cell-surface polysaccharide.     

Efficient in vitro translocation into Escherichia coli membrane vesicles of a protein carrying an uncleavable signal peptide. Characterization of the translocation process

The translocation into Escherichia coli cytoplasmic membrane vesicles of a protein containing an uncleavable signal peptide was studied. The signal peptide cleavage site of the ompF-lpp chimeric protein, a model secretory protein, was changed from Ala-Ala to Phe-Pro through oligonucleotide-directed site-specific mutagenesis of the ompF-lpp gene on a plasmid. The mutant protein was no longer processed by the signal peptidase. When proteinase K treatment was adopted as a probe for protein translocation into inverted membrane vesicles, the mutant protein exhibited rapid and almost complete translocation, most likely due to the lack of premature cleavage of the signal peptide before the translocation. This result also indicates that cleavage of the signal peptide is not required for translocation of the mature domain of the protein. The establishment of an efficient system made it possible to perform precise and quantitative analysis of the translocation process. The translocation was time-dependent, vesicle-dependent, and required ATP and NADH. Translocation into membrane vesicles was also observed with the uncleavable precursor protein purified by means of immunoaffinity chromatography, although the efficiency was appreciably low. The translocation required only ATP and NADH. Addition of the cytosolic fraction did not enhance the translocation.     

MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli

MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymyxin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells.     

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.