Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli

Sensitivity to proteolytic degradation was used to monitor folding of polypeptides in vivo. A correlation between competence for export and lack of stable tertiary structure was established by comparing the kinetics of folding of mutated precursor maltose-binding protein that carries a defective leader peptide with the kinetics of folding of wild-type precursor that is competent for export. It is proposed that during export a kinetic competition exists between productive translocation and folding of precursor intracellularly into a stable conformation that is not compatible with transfer.     

The cell surface expression of group 2 capsular polysaccharides in Escherichia coli: the role of KpsD, RhsA and a multi-protein complex at the pole of the cell

The export of large negatively charged capsular polysaccharides across the outer membrane represents a significant challenge to Gram negative bacteria. In the case of Escherichia coli group 2 capsular polysaccharides, the mechanism of export across the outer membrane was unknown, with no identified candidate outer membrane proteins. In this paper we demonstrate that the KpsD protein, previously believed to be a periplasmic protein, is an outer membrane protein involved in the export of group 2 capsular polysaccharides across the outer membrane. We demonstrate that KpsD and KpsE are located at the poles of the cell and that polysaccharide biosynthesis and export occurs at these polar sites. By in vivo chemical cross-linking and MALDI-TOF-MS analysis we demonstrate the presence of a multi-protein biosynthetic/export complex in which cytoplasmic proteins involved in polysaccharide biosynthesis could be cross-linked to proteins involved in export across the inner and outer membranes. In addition, we show that the RhsA protein, of previously unknown function, could be cross-linked to the complex and that a rhsA mutation reduces K5 biosynthesis suggesting a role for RhsA in coupling biosynthesis and export.     

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.     

Escherichia coli sec mutants accumulate a processed immature form of maltose-binding protein (MBP), a late-phase intermediate in MBP export.

Protein translocation across the Escherichia coli cytoplasmic membrane may consist of several temporally or topographically distinct steps. Although early events in the translocation pathway have been characterized to some extent, the mechanisms responsible for the trans-bilayer movement of a polypeptide are only poorly understood. This article reports on our attempts to dissect the translocation pathway in vivo. A processed form of maltose-binding protein (MBP) was detected in the spheroplasts of secY and secA temperature-sensitive mutant cells that had been pulse-labeled at the permissive temperature (30 degrees C). This species of molecule was found to have an electrophoretic mobility identical to that of the mature MBP, but a considerable fraction of it was inaccessible to externally added protease. It had not attained the protease-resistant conformation characteristically observed for the exported mature protein. The radioactivity associated with this species decreased during chase and was presumably converted into the exported mature form, a process that required energy, probably the proton motive force, as demonstrated by its inhibition by an energy uncoupler. The spheroplast-associated processed form was more predominantly observed in the presence of a low concentration of chloramphenicol. A similar intermediate was also detected for beta-lactamase in wild-type cells. These results suggest that in a late phase of translocation, the bulk of the polypeptide chain can move through the membrane in the absence of the covalently attached leader peptide, and the secA-secY gene products are somehow involved in this process. We termed the processed intermediates processed immature forms.     

Involvement of SecB, a chaperone, in the export of ribose-binding protein.

Ribose-binding protein (RBP) is an exported protein of Escherichia coli that functions in the periplasm. The export of RBP involves the secretion machinery of the cell, consisting of a cytoplasmic protein, SecA, and the integral membrane translocation complex, including SecE and SecY. SecB protein, a chaperone known to mediate the export of some periplasmic and outer membrane proteins, was previously reported not to be involved in RBP translocation even though small amounts of in vitro complexes between SecB and RBP have been detected. In our investigation, it was shown that a dependence on SecB could be demonstrated under conditions in which export was compromised. Species of RBP which carry two mutations, one in the leader that blocks export and a second in the mature protein which partially suppresses the export defect, were shown to be affected by SecB for efficient translocation. Five different changes which suppress the effect of the signal sequence mutation -17LP are all located in the N domain of the tertiary structure of RBP. All species of RBP show similar interaction with SecB. Furthermore, a leaky mutation, -14AE, generated by site-specific mutagenesis causes reduced export in the absence of SecB. These results indicate that SecB can interact with RBP during secretion, although it is not absolutely required under normal circumstances.     

Genetic analysis of the twin arginine translocator secretion pathway in bacteria

The twin arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates translocation of essentially folded proteins across the cytoplasmic membrane. The detailed understanding of the mechanism of protein targeting to the Tat pathway has been hampered by the lack of screening or selection systems suitable for genetic analysis. We report here the development of a highly quantitative protein reporter for genetic analysis of Tat-specific export. Specifically, export via the Tat pathway rescues green fluorescent protein (GFP) fused to an SsrA peptide from degradation by the cytoplasmic proteolytic ClpXP machinery. As a result, cellular fluorescence is determined by the amount of GFP in the periplasmic space. We used the GFP-SsrA reporter to isolate gain-of-function mutants of a Tat-specific leader peptide and for the genetic analysis of the "invariant" signature RR dipeptide motif. Flow cytometric screening of trimethylamine N-oxide reductase (TorA) leader peptide libraries resulted in isolation of six gain-of function mutants that conferred significantly higher steady-state levels of export relative to the wild-type TorA leader. All the gain-of-function mutations occurred within or near the (S/T)RRXFLK consensus motif, highlighting the significance of this region in interactions with the Tat export machinery. Randomization of the consensus RR dipeptide in the TorA leader revealed that a basic side chain (R/K) is required at the first position whereas the second position can also accept Gln and Asn in addition to basic amino acids. This result indicates that twin arginine translocation does not require the presence of an arginine dipeptide within the conserved sequence motif.     

Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation

Accessibility of nascent chains of periplasmic proteins to externally added proteinase K was used as the criterion for translocation of polypeptides across the cytoplasmic membrane of E. coli during the process of export. It is concluded for maltose-binding protein and ribose-binding protein that nascent chains carrying the signal sequence are not accessible to the proteinase while chains that have been matured span the membrane and are degraded. Translocation of polypeptides is a late event relative to extent of elongation, occurring only after maltosebinding protein has reached molecular weight 33,000 (80% of its entire length) and after ribosebinding protein has been fully elongated (molecular weight 29,000). The data presented here are inconsistent with postulated mechanisms of export requiring a strict coupling of translocation to elongation of nascent polypeptide chains. In contrast, the data support the idea that entire domains of polypeptides are transferred after their synthesis. This is the case whether the translocation of a protein is initiated post-translationally or begins before synthesis of the entire protein is completed.     

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.     

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.     

Immediate entrance to the export pathway after synthesis as a requirement for export of the sak gene product in Escherichia coli.

Export through the cytoplasmic membrane and processing of the sak product in Escherichia coli cells were investigated with E. coli strains carrying pTS301, which produce large amounts of staphylokinase at 42 degrees C. High-level synthesis of the sak product caused transient accumulation not only of the staphylokinase precursor (pSAK) but also of the maltose-binding protein and outer membrane protein A precursors. Thus it was concluded that the sak product shares the export pathway with E. coli secreted proteins at least at a certain step. During high-level synthesis of the sak product, a significant amount of the newly synthesized pSAK remained unprocessed after a chase period, possibly causing the observed accumulation of pSAK. Accumulating pSAK did not mature for a long period, whereas the newly synthesized sak product was exclusively detected in the mature form. These results suggest that it is necessary for the sak product to enter the export pathway during or immediately after synthesis to be exported and processed normally.     

Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli

The product of the malE—lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Echerichia coli membrane. The fusion product also inhibited colicin E1 export. Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells. Since colicin E1 contains the internal signal-like sequence [Proc. Natl. Acad. Sci. USA (1982) 79, 2827–2831], these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.     

Processing of preproteins by liposomes bearing leader peptidase

Procoat, the precursor form of M13 coat protein, assembles into sealed liposomes bearing only internally oriented leader peptidase and is processed to yield transmembrane coat protein [Ohno-Iwashita, Y., & Wickner, W. (1983) J. Biol. Chem. 258, 1895-1900]. The precursors of maltose-binding protein and of outer membrane protein A (OmpA) are also processed by these liposomes, showing that these preproteins can at least partially insert across a lipid bilayer. The ability to insert into a bilayer may be a general property of preproteins. The cleavage products, mature OmpA and maltose-binding protein, are not sequestered within the liposomes, suggesting that an additional factor(s) is (are) required for complete translocation. Liposomes were also prepared with leader peptidase in a more physiological, membrane-spanning orientation. These liposomes were also active in the cleavage of externally added procoat, pro-OmpA, and pre maltose-binding protein, though the mature OmpA and maltose-binding protein were still not sequestered within the liposomes. Pretreatment of these liposomes with trypsin cleaved near the amino terminus of the leader peptidase, inactivating the enzyme. The function of this amino-terminal domain, on the opposite side of the membrane from the catalytic domain, is unknown.     

Export of the periplasmic maltose-binding protein ofEscherichia coli

The export of the maltose-binding protein (MBP), themalE gene product, to the periplasm ofEschericha coli cells has been extensively investigated. The isolation of strains synthesizing MalE-LacZ hybrid proteins led to a novel genetic selection for mutants that accumulate export-defective precursor MBP (preMBP) in the cytoplasm. The export defects were subsequently shown to result from alterations in the MBP signal peptide. Analysis of these and a variety of mutants obtained in other ways has provided considerable insight into the requirements for an optimally functional MBP signal peptide. This structure has been shown to have multiple roles in the export process, including promoting entry of preMBP into the export pathway and initiating MBP translocation across the cytoplasmic membrane. The latter has been shown to be a late event relative to synthesis and can occur entirely posttranslationally, even many minutes after the completion of synthesis. Translocation requires that the MBP polypeptide exist in an export-competent conformation that most likely represents an unfolded state that is not inhibitory to membrane transit. The signal peptide contributes to the export competence of preMBP by slowing the rate at which the attached mature moiety folds. In addition, preMBP folding is thought to be further retarded by the binding of a cytoplasmic protein, SecB, to the mature moiety of nascent preMBP. In cells lacking this antifolding factor, MBP export represents a race between delivery of newly synthesized, export-competent preMBP to the translocation machinery in the cytoplasmic membrane and folding of preMBP into an export-incompetent conformation. SecB is one of threeE. coli proteins classified as molecular chaperones by their ability to stabilize precursor proteins for membrane translocation.     

Membrane translocation of mitochondrially coded Cox2p: distinct requirements for export of N and C termini and dependence on the conserved protein Oxa1p.

To study in vivo the export of mitochondrially synthesized protein from the matrix to the intermembrane space, we have fused a synthetic mitochondrial gene, ARG8m, to the Saccharomyces cerevisiae COX2 gene in mitochondrial DNA. The Arg8mp moiety was translocated through the inner membrane when fused to the Cox2p C terminus by a mechanism dependent on topogenic information at least partially contained within the exported Cox2p C-terminal tail. The pre-Cox2p leader peptide did not signal translocation. Export of the Cox2p C-terminal tail, but not the N-terminal tail, was dependent on the inner membrane potential. The mitochondrial export system does not closely resemble the bacterial Sec translocase. However, normal translocation of both exported domains of Cox2p was defective in cells lacking the widely conserved inner membrane protein Oxa1p.     

Role of the TonB amino terminus in energy transduction between membranes.

Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane. TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space. In this report we establish several functions for the hydrophobic amino terminus of TonB. A G-26-->D substitution in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of TonB within the cell envelope. Substitution of the first membrane-spanning domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, suggesting that the amino terminus contains sequence-specific information. Detectable TonB cross-linking to ExbB is also prevented, suggesting that the two proteins interact primarily through their transmembrane domains. In vivo cleavage of the amino terminus of TonB carrying an engineered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density intermediate to those of the cytoplasmic and outer membranes, and (iii) cross-linking to ExbB. In contrast, the amino terminus is not required for cross-linking to other proteins with which TonB can form complexes, including FepA. Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates with the cytoplasmic membrane. TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane.     

Export incompatibility of N-terminal basic residues in a mature polypeptide of Escherichia coli can be alleviated by optimising the signal peptide

Summary Export of the outer membrane protein, OmpA, across the cytoplasmic membrane of Escherichia coli was severely inhibited by the presence of two, three, four or six additional basic residues at the N-terminus of the mature polypeptide, but not by three similarily positioned acidic residues. Because a few bacterial proteins do possess basic residues close to the leader peptidase cleavage site and because the type of inhibition described here could pose problems in the construction of hybrid secretory proteins, we also studied means of alleviating this form of export incompatibility. Inhibition was abolished when basic residues were preceded by acidic ones. Also, the processing rates of the mutants with two or six basic residues could be partially restored by increasing the length of the hydrophobic core of the signal peptide. Taking this as a precedent, it is suggested that the structure of the signal peptide is an important feature for maintenance of a reasonable rate of translocation of those exported proteins which possess basic residue(s) at the N-terminus of the mature polypeptide.     

In vitro insertion of leader peptidase into Escherichia coli membrane vesicles.

Leader peptidase is an integral protein of the Escherichia coli cytoplasmic membrane whose topology is known. We have taken advantage of this knowledge and available mutants of this enzyme to develop a genetic test for a cell-free protein translocation reaction. We report that leader peptidase inserted into inverted plasma membrane vesicles in its correct transmembrane orientation. We have examined the in vitro membrane assembly characteristics of a variety of leader peptidase mutants and found that domains required for insertion in vivo are also necessary for insertion in vitro. These data demonstrate the physiological validity of the in vitro insertion reaction and strengthen the use of this in vitro protein translocation reaction for the dissection of this complex sorting pathway.     

Demonstration in vivo that interaction of maltose-binding protein with SecB is determined by a kinetic partitioning.

An early step in the export of maltose-binding protein to the periplasm is interaction with the molecular chaperone SecB. We demonstrate that binding to SecB in vivo is determined by a kinetic partitioning between the folding of maltose-binding protein to its native state and its association with SecB. A complex of SecB and a species of maltose-binding protein that folds slowly is shown to be longer-lived than a complex of the wild-type maltose-binding protein and SecB. In addition, we show that incomplete nascent chains, which are unable to fold, remain complexed with SecB.     

Export and processing of MalE-LacZ hybrid proteins in Escherichia coli

Five classes of MalE-LacZ hybrid proteins have previously been characterized. These proteins differ in the amount of the maltose-binding protein (MBP) that is attached to beta-galactosidase. Although none of these proteins is secreted into the periplasm, the four larger classes of hybrid proteins, those that include an intact MBP signal peptide, are inserted into the cytoplasmic membrane, suggesting that the secretion process has at least been initiated. In this study, we demonstrated that some portion of the four larger hybrid proteins can be translocated across the cytoplasmic membrane, thus permitting processing of the signal peptide. We have found that hybrid proteins that include only a small portion of the mature MBP are inefficiently recognized as exported proteins, and translocation and processing of these appear to be relatively slow, posttranslational events. In marked contrast, hybrid proteins that include a substantial portion of the mature MBP are efficiently recognized, and translocation and processing of these occur very rapidly, possibly cotranslationally. Our results complement other studies and very strongly suggest a role for the mature MBP in the export process.     

Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process.

The requirements for the translocation of prolipoprotein into membrane vesicles were examined in an in vitro system. As measured by the eventual modification and processing of the prolipoprotein to form mature lipoprotein, the overall translocation process was found to require ATP hydrolysis, the presence of some heat-labile soluble cytoplasmic translocation factors, and the function of a cytoplasmic membrane protein, SecY/PrlA. However, the initial step of complete insertion of prolipoprotein into the membrane vesicles occurred without apparent requirements of a nucleotide, cytoplasmic translocation factors, or a functional SecY/PrlA membrane protein. Immunopurified prolipoprotein spontaneously inserted into membrane vesicles at elevated temperatures and required ATP and cytoplasmic translocation factors to form mature lipoprotein. The prolipoprotein inserted most efficiently into liposomes made of negatively charged phospholipids, indicating the importance of phospholipids in protein translocation. These results suggest that ATP hydrolysis and the actions of both cytoplasmic translocation factors and a functional SecY/PrlA membrane protein occur at a step(s) after the insertion of the precursors into membrane vesicles. The initial step of spontaneous insertion of prolipoprotein into membranes is in good agreement with membrane trigger hypothesis proposed by W. Wickner (Annu. Rev. Biochem. 48:23-45, 1979) and the helical hairpin hypothesis proposed by D. M. Engleman and T. A. Steitz (Cell 23:411-422, 1981).