ROLE OF A POTENTIAL ENDOPLASMIC-RETICULUM RETENTION SEQUENCE (RDEL) AND THE GOLGI-COMPLEX IN THE CYTOTONIC ACTIVITY OF ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN

Recent experimental evidence indicates that Escherichia coli heat-labi le enterotoxin and the closely related cholera toxin gain access to in tracellular target substrates through a brefeldin A-sensitive pathway that may involve retrograde transport through the Golgi-endoplasmic re ticulum network. The A subunits of both toxins possess a carboxy-termi nal tetrapeptide sequence (KDEL in cholera toxin and RDEL in the heat- labile enterotoxins) that is known to mediate the retention of eukaryo tic proteins in the endoplasmic reticulum. To investigate the potentia l role of the RDEL sequence in the toxic activity of the heat-labile e nterotoxin we constructed mutant analogues of the toxin containing sin gle substitutions (RDGL and RDEV) or a reversed sequence (LEDR). The s ingle substitutions had little effect on Chinese hamster ovary cell el ongation or the ability to stimulate cAMP accumulation in Caco-2 cells . Reversal of the sequence reduced the ability of the toxin to increas e cAMP levels in Caco-5 cells by approximately 60% and decreased the a bility to elicit elongation of Chinese hamster ovary cells. The effect s of the heat-labile enterotoxin were not diminished in a mutant Chine se hamster ovary cell line (V.24.1) that belongs to the End4 complemen tation group and possesses a temperature-sensitive block in secretion that correlates directly with the disappearance of the Golgi stacks. C ollectively, these findings suggest that the brefeldin A-sensitive pro cess involved in intoxication by the heat-labile enterotoxin does not involve RDEL-dependent retrograde transport of the A subunit through t he Golgi-endoplasmic reticulum complex. The results are more consisten t with a model of internalization involving translocation of the A sub unit from an endosomal or a trans-Golgi network compartment.