TARGETING OF CHOLERA-TOXIN AND ESCHERICHIA-COLI HEAT-LABILE TOXIN IN POLARIZED EPITHELIA - ROLE OF COOH-TERMINAL KDEL

Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) el icit a secretory response from intestinal epithelia by binding apical receptors (ganglioside G(M1)) and subsequently activating basolateral effecters (adenylate cyclase), We have recently proposed that signal t ransduction in polarized cells may require transcytosis of toxin-conta ining membranes (Lencer, W, I., G. Strohmeier, S. Moe, S, L, Carlson, C, T. Constable, and J, L,, Madara, 1995. Proc. Natl. Acad. Sci. USA. 92:10094-10098). Targeting of CT into this pathway depends initially o n binding of toxin B subunits to G(M1) at the cell. surface, The anato mical compartments in which subsequent steps of CT processing occur ar e less clearly defined, However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT), Thus if the KD EL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied. To test this idea, recombinant wild- type (wt) and mutant CT and LT were prepared. The COOH-terminal KDEL s equence in CT was replaced by seven unrelated amino acids: LEDERAS. In LT, a single point mutation replacing leucine with valine in RDEL was made. Wt and mutant toxins displayed similar enzymatic activities and binding affinities to G(M1) immobilized on plastic. Biologic activity of recombinant toxins was assessed as a Cl- secretory response elicit ed from the polarized human epithelial cell line T84 using standard el ectrophysiologic techniques, Mutations in K(R)DEL of both CT and LT de layed the time course of toxin-induced Cl- secretion. At T1/2, dose de pendencies for K(R)DEL-mutant toxins were increased greater than or eq ual to 10-fold. KDEL-mutants displayed differentially greater temperat ure sensitivity. In direct concordance with a slower rate of signal tr ansduction, KDEL-mutants were trafficked to the basolateral membrane m ore slowly than wt CT (assessed by selective cell surface biotinylatio n as transcytosis of B subunit). Mutation in K(R)DEL had no effect on the rate of toxin endocytosis, These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that bo th toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.