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

Abstract

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.

Folded HasA inhibits its own secretion through its ABC exporter

“…Only unfolded proteins can be translocated to the periplasm by the Sec machinery. N‐terminal signal sequences and cytoplasmic chaperones ensure that proteins remain competent for translocation (Rosemond et al ., 1994; Wild et al ., 1996; Randall et al ., 1998). In contrast, the Tat pathway translocates proteins that are at least partially folded and often carrying a cofactor (Berks et al ., 2000).…”

Escherichia coli SecB stimulates export without maintaining export competence of ribose-binding protein signal sequence mutants

“…In vitro studies with purified preRBP demonstrate that pre-RBP folds rapidly into an export-incompetent conformation and that SecB has little effect on pre-RBP folding and export competence (8). Taken together, the results of Kim et al (21), Cox Rosemond et al (8), and this study suggest that maintaining pre-RBP export competence is unlikely to be the mechanism by which SecB promotes RBP export. In fact, reversible SecB binding may promote pre-RBP folding (21).…”

“…However, in the presence of a mutation in the early mature region of RBP, which slows down its folding, translocation could be observed at later time points (21). In vitro studies with purified preRBP demonstrate that pre-RBP folds rapidly into an export-incompetent conformation and that SecB has little effect on pre-RBP folding and export competence (8). Taken together, the results of Kim et al (21), Cox Rosemond et al (8), and this study suggest that maintaining pre-RBP export competence is unlikely to be the mechanism by which SecB promotes RBP export.…”