Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro.

Abstract

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...

Mutational Alterations in the Homotetrameric Chaperone SecB That Implicate the Structure as Dimer of Dimers

“…SecB was purified from strain BL21(DE3) pJW25 (6). SecBL75Q, SecBE77K, and SecBF74I were purified from strain CK2212 (BL21 (DE3) secB::Tn5 srl::Tn10 recA) containing two plasmids.…”

“…Lysates-Soluble lysates were prepared (as above except that the growth temperature was 35°C) from the following strains: CK1953 (10), which carries the secB::Tn5 mutation and is secBnull; BL21(DE3) pJW25 (6), which carries the wild-type secB on the chromosome as well as on the plasmid; and CK2212 with two plasmids both containing either secBC76Y or secBV78F. One of the two plasmids carries the secB variant gene under its natural promoter, and the other plasmid carries the secB variant gene under control of the T7 promoter.…”

Electrospray mass spectrometric investigation of the chaperone SecB

“…The initial evidence that SecB is oligomeric came from analysis by size-exclusion chromatography and electrophoresis of the native protein through polyacrylamide gels. However, a range of molecular weights have been reported: 64 kDa (Watanabe & Blobel, 1989), 79 kDa (Weiss et al, 1988), 90 kDa , and 115 kDa (Weiss et al, 1988), and thus the oligomeric state has not been determined definitively. The range of values obtained would be explained if SecB deviated from the ideal spherical structure because the techniques used base the determination of molecular weight on the separation of molecules according to their radii of gyration.…”

In‐vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE

“…SecB is one of the cytosolic factors which is required for the translocation of a subset of precursor proteins (Kumamoto and Beckwith, 1985). In-vitro studies with SecB purified from an over-producing (Weiss et al, 1988;Kumamoto et al, 1989) or non-over-producing (Watanabe and Blobel, 1989a) strain, showed that it promotes in a tetrameric (Watanabe and Blobel, 1989a) or an oligomeric (Weiss et al, 1988) form the translocation of in-vitrosynthesized precursor proteins. It has also been shown that SecB stabilizes the translocation-competent state of the purified precursors of the E. coli outer membrane proteins PhoE (prePhoE) and OmpA (preOmpA) diluted from concentrated urea solutions in vitro (Kusters et al, 1989; Lecker et al., 1989).…”