Molecular Microbiology (1995) 16(4). 789-800 Role of a potential endoplasmic reticulum retention sequence (RDEL) and the Golgi complex in the cytotonic activity of Escherichia coli heat-labile enterotoxin Witold Cieplak, Jr,* Ronald J. Messer, Michael E. Konkel and Christopher C. R. Grant Laboratory of Intracellular Parasites. Roci<y Mountain Laboratories. National Institute of Allergy and Infectious Diseases. National institutes of Health. Hamilton. Montana 59840. USA. Summary Recent experimental evidence indicates that Escheri- chia coli heat-labile enterotoxin and the closely related cholera toxin gain access to intracellular target sub- strates through a brefeldin A-sensitive pathway that may involve retrograde transport through the Golgi- endoplasmic reticulum network. The A subunits of both toxins possess a carboxy-terminal tetrapeptide sequence (KDEL in cholera toxin and RDEL in the heat-labile enterotoxins) that Is known to mediate the retention of eukaryotic proteins in the endoplasmic reticuium. To investigate the potential role of the RDEL sequence in the toxic activity of the heat-labile entero- toxin we constructed mutant analogues of the toxin containing single substitutions (RDGL and RDEV) or a reversed sequence (LEDR). The single substitutions had little effect on Chinese hamster ovary cell elonga- tion or the ability to stimulate cAMP accumulation in Caco-2 cells. Reversal of the sequence reduced the ability of the toxin to increase cAMP levels in Caco-2 cells by approximately 60% and decreased the ability to elicit elongation of Chinese hamster ovary cells. The effects of the heat-labile enterotoxin were not diminished in a mutant Chinese hamster ovary cell line (V.24.1) that belongs to the End4 complementa- tion group and possesses a temperature-sensitive block in secretion that correlates directly with the disappearance of the Golgi stacks. Collectively, these findings suggest that the brefeldin A-sensitive process involved in intoxication by the heat-labile enterotoxin does not involve RDEL-dependent retrograde trans- port of the A subunit through the Golgi-endoplasmic reticulum complex. The results are more consistent Received 27 April. 1994: revised 19 January. 1995, accepted 26 Jan- uary, 1995. *For correspondence. E-mail WC@RML.NIAID.NIH.GOV; Tel. (406) 363 9262; Fax (406) 363 9253. with a model of internalization involving translocation of the A subunit from an endosomal or a frans-Golgi network compartment. Introduction Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) are closely related toxins that exert their effects through the ADP-ribosylation of regulatory G proteins (Gg,) of the adenylate cyctase complex in eukaryotic cell mem- branes (reviewed in Spangler, 1992). ADP-ribosylation of the subunits distiipts normal regulation of adenylate cyclase activity and results in increases in intracellular cAMP. Both toxins are composed of enzymatically active A subunits {M, 30000) and a pentamer composed of identical B subunits (Mr 11 000). The B homopentamer binds to gangliosides on the surface of mammalian cells (Fishman, 1982). The A subunit of LT is a single-chain molecule that is activated by limited trypsinolysis and reduction in vitro (Lee et ai. 1991). Trypsin-mediated cleavage occurs at an arginine residue (Arg-192), which lies in a region that is subtended by the sole disulphide bond in the A subunit, and results in the formation of the enzymatically active Al fragment (M, 23000) and a smaller fragment, termed A2. derived from the carboxy-terminal portion of the molecule (Grant et ai. 1994). In addition to cleavage and reduction, small GTP-binding proteins known as ADP-ribosylation factors (ARFs) are also required for full enzymatic activation of both LT and CT in vitro (Kahn and Gilman, 1986; Moss and Vaughn, 1991; Lee etai. 1991)- While the general enzymatic and binding properties of these toxins are reasonably well understood, there is little information concerning the mechanism by which the enzy- matically active A or Al subunits gain access to Gs-, on the cytoplasmic face of the plasma membrane or other intra- cellular membranous compartments. Several models of toxin internalization based on various types of evidence have been proposed, and involve either direct trans- location of the A subunit across the plasma membrane (Wisnieski and Bramhall. 1981; Tomasi and Montecucco, 1981) or translocation from an endosomal compartment after endocytosis (Tran et ai. 1987; Janicot et ai. 1991; Lencer ef ai. 1993). Recent evidence has led to the suggestion that the A subunits of CT and LT may be processed and gain access to the cytoplasm via a route 1995 Blackwell ScierKe Ltd