P J Bassford

The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein

Evidence is presented that the E. coli secB gene encodes a soluble protein that interacts with the mature region of the precursor maltose-binding protein (MBP), and promotes MBP export by preventing premature folding of the newly synthesized polypeptide into an export-incompetent form. The interaction of SecB with MBP was indicated by the finding that synthesis of various export-defective MBP species interfered with normal protein export by limiting SecB availability. The antifolding activity of SecB was demonstrated by the following: the defect in MBP export in SecB- cells was suppressed by mutational alterations affecting MBP folding; export of a mutant MBP that is accomplished in a strictly posttranslational mode was totally blocked in SecB- cells; and the rate of folding of wild-type MBP synthesized in vitro was found to be accelerated when SecB was absent and greatly retarded when excess SecB was present.

Protein localization in E. coli: Is there a common step in the secretion of periplasmic and outer-membrane proteins?

An E. coli strain carrying a fusion of the MalE and lacZ genes is induced for the synthesis of a hybrid protein, consisting of the N-terminal part of the maltose-binding protein and the enzymatically active C-terminal part of beta-galactosidase, by addition of maltose to cells. The secretion of the protein is initiated by the signal peptide attached to the N terminus of the maltose-binding protein sequence, but is not completed, presumably because the beta-galactosidase moiety of the hybrid protein interferes with the passage of the polypeptide through the cytoplasmic membrane. Thus the protein becomes stuck to the cytoplasmic membrane. Under such conditions, periplasmic proteins, including maltose-binding protein (encoded by the malE gene) and alkaline phosphatase, and the major outer-membrane proteins, including OmpF, OmpA and probably lipoprotein, are synthesized as precursor forms with unprocessed signal sequences. This effect is observed within 15 min after high levels of induction are achieved. The simplest explanation for these results and those of pulse-chase experiments is that specific sites in the cytoplasmic membrane become progressively occupied by the hybrid protein, resulting in an inhibition of normal localization and processing of periplasmic and outer-membrane proteins. These results suggest that most of the periplasmic and outer-membrane proteins share a common step in localization before the polypeptide becomes accessible to the processing enzyme. If this interpretation is correct, we can estimate that an E. coli cell has roughly 2 x 10(4) such sites in the cytoplasmic membrane. A system is described for detecting the precursor of any exported protein.

A signal sequence is not required for protein export in prlA mutants of Escherichia coli

The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose‐binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal‐sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal‐sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.

Escherichia coli mutants accumulating the precursor of a secreted protein in the cytoplasm

The maltose binding protein of Escherichia coli is secreted into the external periplasmic compartment of the cell. This article describes a selection procedure for the isolation of mutants which fail to export this protein. These mutants probably result from alterations in the amino terminal ‘signal sequence’, causing the maltose binding protein produced to accumulate in the cytoplasm in its precursor form.

Intragenic suppressor mutations that restore export of maltose binding protein with a truncated signal peptide

A deletion mutation, malE delta 12-18, removes seven residues from the hydrophobic core of the maltose binding protein (MBP) signal peptide and thus prevents secretion of this protein to the periplasm of E. coli. Intragenic suppressor mutations of malE delta 12-18 have been obtained, some highly efficient in their ability to restore proper MBP export. Twelve independently isolated suppressors represent six unique mutational events. Five result in alterations within the MBP signal peptide; one changes the amino acid at residue 19 of the mature MBP. Analysis of these suppressors indicates that the length of the hydrophobic core is a major determinant of signal peptide function. The experiments further suggest that the hydrophobic core region serves primarily a structural role in mediating protein secretion, and that other sequences outside of this region may be responsible for providing the initial recognition of the MBP nascent chain as a secreted protein.

In vitro assay to demonstrate high-level erythromycin resistance of a clinical isolate of Treponema pallidum.

We have previously demonstrated that cells of Treponema pallidum freshly extracted from infected rabbit testes can be intrinsically radiolabeled with [35 S]methionine to very high specific activities. In this study we used the inhibition of [35 S]methionine incorporation into trichloroacetic acid-precipitable protein in vitro as an assay to test the susceptibilities of three different pathogenic treponemal strains to various antibiotics. In general, the results correlated very well with the known efficacies of these antibiotics in treating human patients with syphilis. One of the strains tested, however, a clinical isolate of T. pallidum designated street strain 14, was found to exhibit high-level resistance to erythromycin and a closely related macrolide, roxithromycin (RU 965). Street strain 14 was originally isolated from a human patient with active secondary syphilis who failed to respond to erythromycin therapy. Thus, our results indicate that an erythromycin-resistant strain of T. pallidum can be responsible for erythromycin treatment failure. In addition, street strain 14 treponemes were found to be generally less susceptible by this assay to a variety of antibiotics than were treponemes of the T. pallidum Nichols strain. These findings suggest that the outer envelope of street strain 14 treponemes may be generally less permeable to antibiotics than is that of Nichols strain treponemes. Images

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.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.

Ability of MBP or RBP signal peptides to influence folding and in vitro translocation of wild-type and hybrid precursors

Maltose‐binding protein (MBP), whose export in E. coli is dependent upon the chaperone SecB, and ribose‐binding protein (RBP), whose export is SecB‐independent, have been used to generate hybrid secretory proteins. Here, in vitro techniques were used to analyze MBP, RBP, RBP‐MBP (RBP signal and MBP mature), and MBP‐RBP (MBP signal and RBP mature). In protease‐protection experiments, RBP folded considerably faster than MBP, RBP‐MBP, or MBP‐RBP. Only the folding properties of proteins containing the MBP mature moiety were influenced by SecB. In post‐translational translocation assays, MBP exhibited the highest translocation efficiency. The hybrids RBP‐MBP and MBP‐RBP showed intermediate levels, and RBP translocation was not detected in these assays. These experiments demonstrate the influence of the signal peptide in determining folding properties and translocation efficiency of precursor secretory proteins.