Antifolding Activity of the SecB Chaperone Is Essential for Secretion of HasA, a Quickly Folding ABC Pathway Substrate

Abstract

We have previously shown that SecB, the ATP-independent chaperone of the Sec pathway, is required for the secretion of the HasA hemophore from Serratia marcescens via its type I secretion pathway, both in the reconstituted system in Escherichia coli and in the original host. The refolding of apo-HasA after denaturation with guanidine HCl was followed by stopped-flow measurements of fluorescence of its single tryptophan, both in the absence and presence of SecB. In the absence of SecB, HasA folds very quickly with one main phase (45 s ؊1 ) accounting for 92% of the signal. SecB considerably slows down HasA folding. At stoichiometric amounts of SecB and HasA, a single phase (0.014 s ؊1 ) of refolding is observed. Two double point mutants of HasA were made, abolishing two hydrogen bonds between N-terminal and C-terminal side chain residues. In both cases, the mutants essentially maintained the same secondary and tertiary structure as wild-type HasA and were fully functional. Refolding of both mutants was much slower than that of wild-type HasA and they were secreted essentially independently of SecB. We conclude that SecB has mainly an antifolding function in the HasA ABC secretion pathway.

Mutations in HlyD, Part of the Type 1 Translocator for Hemolysin Secretion, Affect the Folding of the Secreted Toxin

“…This is supported by recent studies of HasA, a small 21-kDa protein that folds in the cytoplasm in the absence of the chaperone SecB and cannot be translocated (11,53). Consequently, folding of such type 1 proteins, including HlyA, must take place on the cell surface.…”

“…Recent studies have provided further evidence that type I secretion involves translocation of unfolded proteins. These studies have shown that the secretion of a small 19-kDa protein, HasA, in Serratia marcescens is dependent upon the chaperone SecB and cannot be transported if allowed to fold in the periplasm (42,53).…”

Multiple Signals Direct the Assembly and Function of a Type 1 Secretion System

“…All plasmids used in this study are listed in Table 1. pAM238-HasA and derivative plasmids carrying HasA E148A, D167A or HasA mutant forms with pentapeptide insertions were described elsewhere (29,30,45). hasA alleles were amplified by PCR from the appropriate plasmid templates with the forward primer 5Ј-TTCACCATGGCATTTTCAGTCAATTAT GAC-3Ј and the reverse primer 5Ј-CGACTCTAGATCACGCCGTCGCCGCC GCCA-3Ј.…”

“…Secretion of hemophores depends on the presence of the general chaperone SecB (13). SecB holds premade HasA molecules in an unfolded (or loosely folded) conformation compatible with secretion but does not function to deliver HasA to HasD (35,45). However, we have previously shown that delayed expression of HasDE causes HasA to fold in the cytoplasm, accumulate, and inhibit the secretion of newly syn-thesized molecules, indicating that synthesis and secretion must be coupled (11).…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…72,2008 BACTERIAL ATP-BINDING CASSETTE SYSTEMS 341 on May 9, 2018 by guest http://mmbr.asm.org/ folded proteins pass through ABC proteins in the cytoplasmic membrane. SecB, a cytoplasmic chaperone that functions to slow the rate of folding of proteins destined for secretion by the general secretory pathway, slows the folding of HasA and is required for secretion of HasA, suggesting that HasA must be unfolded to be transported (112,418,529). If HasA is allowed to fold in the cytoplasm, it inhibits secretion of newly synthesized molecules, suggesting that the folded protein retains affinity for the transporter but is not competent for transport (109).…”