Variant forms of SecB with substitutions of aminoacyl residues in the region from 74 to 80 were analyzed with respect to their ability to bind a physiological ligand, precursor galactose-binding protein, and to their oligomeric states. SecBL75Q and SecBE77K are tetramers with affinity for ligand indistinguishable from that of the wild-type SecB, and thus the export defect exhibited by strains producing these variants must result from an effect on interactions between SecB and other components. SecBF74I is tetrameric but binds ligand with a lower affinity. Substitutions at positions 76, 78, and 80 cause a shift in the equilibrium so that the SecB tetramer dissociates into dimers. We conclude that the tetramer is a dimer of dimers and that the residues Cys 76 , Val 78 , and Gln 80 must be involved either directly or indirectly in forming the interface between dimers. These variant species are defective in binding ligand; however, because their oligomeric state is altered no conclusion can be drawn concerning the direct role of these residues in ligand binding.SecB is a chaperone from Escherichia coli involved in the facilitation of export of proteins from the cytoplasm to the outer membrane, which lies beyond the cytoplasmic membrane, and to the periplasm, the aqueous space between the two membranes. Like all chaperones it binds its ligands by virtue of their non-native state. The binding is highly selective for unstructured proteins; however, no motif among the ligands has been found that might serve as a recognition element for SecB (1, 2). Mutant strains of E. coli that produce altered species of SecB and are defective in protein export have been isolated by Kumamoto and co-workers (3). Analyses of complexes between SecB and a ligand, precursor maltose-binding protein, isolated by co-immunoprecipitation from these mutant strains led to the proposal that the aminoacyl residues in the region from 74 to 80 at the even numbered positions, i.e. Phe had no effect on the efficiency of co-immunoprecipitation of SecB and ligand; thus, it seems that the effect on export observed in vivo is the result of a defective interaction with other components of the export pathway. Here we have further investigated the nature of the defects exhibited by the altered species of SecB and conclude that the region implicated in binding of the precursors is crucial in maintaining the quaternary structure of SecB. SecB is a tetramer of identical subunits, but the species with substitutions at the positions 76, 78, and 80 exist as dimers. We conclude that these residues must be involved either directly or indirectly in forming the interface of dimers that normally associate to form tetramers and that substitutions at these positions shift the equilibrium so that the tetramer dissociates. EXPERIMENTAL PROCEDURESProtein Purification-Mature galactose-binding protein and precursor galactose-binding protein were purified as described (4, 5). SecB was purified from strain BL21(DE3) pJW25 (6). SecBL75Q, SecBE77K, and SecBF74I were purified from s...

The secretion of protein directly into the extracellular medium by Bacillus amyloliquefaciens, a gram-positive bacterium, was shown to be dependent on proton motive force. When the electrochemical membrane potential gradient of protons was dissipated either by unco plers or by valinomycin in combination with K+, a precursor form of a-aamylase accumulated on the cellular membrane. The proton motive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition of export was not the result of decreased ATP concentration.Proton motive force is essential to the translocation of polypeptides through membranes in some but not all systems. Export of protein in Escherichia coli, a gram-bacteria bacterium, requires an electrochemical membrane potential, as does import of protein to the inner compartments of mitochondria. However, passage of protein through the membrane of the rough endoplasmic reticulum during secretion is independent of energy (L. L. Randall, Methods Enzymol., in press). Whereas the lumen of the endoplasmic reticulum is bounded by a single membrane, gram-negative bacteria and mitochondria are each surrountded by two membranes. Thus, the energy requirement in the latter two systems might be related to that common structural feature. If this were the case, then a gram-positive bacterium, bounded by a single membrane analogous to the rough endoplasmic reticulum, would not require energy. Therefore, we began investigations of the energetics of export in Bacillus amyloliquefaciens. We conclude that the energy requirement seen in mitochondria and E. coli is not likely to be related to the presence of a double membrane, because, as we report here, B. amyloliquefaciens requires proton motive force to secrete a-amylase through a single membrane.MATERIALS AND METHODS Chemicals.[35S]methionine (approximately 1,000 Ci/ mmol) was purchased from New England Nuclear Corp., and Staphylococcus aureaus V8 protease was obtained from Miles Laboratories, Inc. Chloramphenicol, phenylmethylsulfonyl fluoride (PMSF), lysozyme, firefly lantern extract, valinomycin, and oa-amylase were purchased from Sigma Chemical Co. SF 6847 was a gift from Evert Bakker.Strains and growth conditions. B. amyloliquefaciens SYC 5085 was grown at 37°C with shaking in M9 minimal medium (9) containing 18 amino acids (all except methionine and cysteine) at 1 mM and 33 mM glucose.Pulse-labeling. Exponentially growing cells were pulselabeled at a density of 4 x 108 cells per ml. The cell culture (1 ml) was labeled for 30 s with 40 ,uCi of [35S]methionine (pulse-label), and then incorporation of label was terminated by the addition of unlabeled methionine to a concentration of 0.1 mM. The culture was incubated for an additional 60 s (nonradioactive chase) and then poured onto 0.5 ml of frozen 10 mM Tris-acetate (pH 7.8)-0.1 mM PMSF. In experiments involving labeling in the presence of uncouplers, the cultures * Corresponding author. were incubated in the presence of carbonyl cyanide inchlorophenylhydrazo...