Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae.

Abstract

The secretion of enterotoxin by Vibrio cholerae is'punctuated by the transient entry of the toxin subunits into the periplasm. In this paper, we show that the subunits oligomerize into an assembled holotoxin within the periplasm prior to their secretion across the outer membrane. The rate of toxin assembly was studied by pulse-labeling cells with [355]-methionine and then monitoring the turnover of radiolabeled subunits as they assembled withi the periplasm. The subunits entered the periplasm as monomers and assembled into oligomers with a half-time of -1 min. Since assembly was a rapid event compared to the rate of toxin efflux from the periplasm, which had a half-time of 13 nin, we conclude that all of the subunits that pass through the periplasm assemble before they traverse the outer membrane. The average concentration of subunit monomers and assembled holotoxin within the periplasm was calculated to be -20 and =260 ,ug/ml, respectively. This indicates that the periplasm is a suitably concentrated milieu where spontaneous toxin assembly can occur. Our findings suggest that protein movement across bacterial outer membranes, in apparent contrast to export across other biological membranes, involves translocation of polypeptides that have already folded into tertiary and even quaternary conformations.The movement of proteins across membranes is a fundamental characteristic of living cells. It plays an obligatory role in the replenishment and growth of membrane systems and cell envelopes, in organelle biogenesis, and in the secretion of, for example, protein hormones, immunoglobulins, and extracellular enzymes (for reviews, see refs. 1-4). A number of recent reports on protein import into mitochondria, and export of proteins across the cytoplasmic membrane of Escherichia coli have implied that nonnative, disordered, or unfolded protein states are needed to ensure successful translocation (5,6). In addition, several research groups, studying the secretion ofproteins into the endoplasmic reticulum (ER) and from the ER to the Golgi complex in eukaryotic cells, have found that protein folding, and in particular subunit oligomerization, are intraluminal processes occurring after translocation across the ER membrane (7-10). Thus, proteins that are translocated across ER and mitochondrial and bacterial cytoplasmic membranes would seem to share a common feature of initiating their export as unfolded polypeptides that develop native tertiary or quaternary conformations only after trAnslocation has occurred (5-12).

XpsD, an Outer Membrane Protein Required for Protein Secretion by Xanthomonas campestris pv. campestris, Forms a Multimer

“…In this study we provide evidences for the first time to show that an OM component of one of the terminal branches of general secretion pathway (5) probably forms stable complex of a minimal 12-mer. An OM secretion channel is anticipated for secreted proteins that are translocated across OM as disulfide-bonded molecules (35)(36)(37)(38)(39).…”

Protein secretion and the pathogenesis of bacterial infections

“…V. cholerae secrete cholera toxin, which is composed of CtxA and CtxB subunits . Mature CtxB assembles into a pentameric ring structure within the bacterial periplasm (Hirst and Holmgren 1987) and associates with the C-terminal domain of mature CtxA to form cholera toxin, CtxA:CtxB 5 Lonnroth and Holmgren 1973;Finkelstein et al 1974;Heyningen 1974). CtxA forms an intramolecular disulfide bond and is proteolytically processed Lonnroth and Holmgren 1973;Heyningen 1974).…”

Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway

“…Although unusual, such a dual-energy requirement for protein transport through a single membrane is not unprecedented; for example, transport of precursor proteins through the cytoplasmic membrane by the first part (Sec) of the GSP requires both ATP hydrolysis (by the SecA protein) and the pmf (Geller, 1991;Pugsley, 1993a). Alternatively, protein translocation across the outer membrane could be driven by diffusion down the concentration gradient between the periplasmic and extracellular pools of the exoprotein (Hirst and Holmgren, 1987).…”