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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.

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The efficient export of a subset ofEscherichia coli envelope proteins is dependent upon the product of the secB gene. Previous studies indicated that SecB promotes the export of the periplasmic maltose-binding protein (MBP) by preventing premature folding of the precursor MBP in the cytoplasm into an export-incompetent form. In this study, SecB has been purified to homogeneity and shown to be a soluble, cytoplasmic, multimeric protein composed of identical 17-kDa subunits. SecB was required for efficient in vitro translocation of MBP into inverted membrane vesicles. The addition of purified SecB to an in vitro system prepared from SecB-cells significantly enhanced MBP translocation. The purified protein also quantitatively retarded folding of precursor MBP into a stable, protease-resistant conformation in the absence of membranes. Finally, the inclusion of excess purified SecB in a SecB+ in vitro system significantly prolonged the time in which precursor MBP remained competent for posttranslational import into membrane vesicles.A number of components of the protein export machinery of Escherichia coli have been identified by genetic approaches [reviewed by Oliver (1)] and by biochemical approaches using in vitro protein translocating systems (2-4). The export of a subset of envelope proteins, including the periplasmic maltose-binding protein (MBP), is adversely affected by mutations in secB, a nonessential gene that maps near min 81 on the E. coli chromosome (5, 6). We have presented (7) evidence that the SecB protein functions as an antifolding factor that specifically interacts with the mature region of the precursor MBP (pre-MBP) to prevent its premature folding in the cytoplasm into a translocation-incompetent form. Kumamoto and Gannon (8) reached a similar conclusion by using a different experimental approach. Thus a specific biochemical function was assigned to the product of an E. coli sec gene, which complemented earlier work by Randall and Hardy (9) correlating the folding of pre-MBP into its stable tertiary structure in the cytoplasm with the loss ofexport competence. The essential role of protein conformation during membrane translocation has been recognized in eukaryotic cells as well (10). For example, unfolding factors that facilitate translocation of proteins into the endoplasmic reticulum and mitochondria have been described (11,12).An understanding ofprotein export in E. coli will be greatly aided by the purification and characterization of the individual components that mediate this process and eventual reconstitution of a complete protein translocating system in vitro. This study was undertaken to demonstrate the SecB dependence of MBP translocation in vitro and to purify biologically active SecB capable of interacting with newly synthesized pre-MBP to retard its folding and promote its import into membrane vesicles. MATERIALS AND METHODSIn Vitro MBP Synthesis and Translocation. MBP was synthesized in vitro by using a coupled E. coli transcriptiontranslation system, in the presence o...

The maltose binding protein of Escherichia coli is secreted into the external periplasmic compartment of the cell by virtue of an amino-terminal signal sequence. Using DNA sequencing, we have determined the precise nature of mutations in the signal sequence which prevent the export of the maltose binding protein, causing it to accumulate in the cytoplasm in its precursor form. In most cases, the change of a single hydrophobic or uncharged amino acid to a charged amino acid within the signal sequence is sufficient to block the secretion process.