ADDITION OF AN ENDOPLASMIC-RETICULUM RETRIEVAL SEQUENCE TO RICIN-A CHAIN SIGNIFICANTLY INCREASES ITS CYTOTOXICITY TO MAMMALIAN-CELLS

An Escherichia coli expression system was used to produce recombinant ricin A chain (RTA) and RTA modified either by the addition of a carbo xyl-terminal endoplasmic reticulum retrieval sequence Lys-Asp-Glu-Leu (RTAKDEL) or a nonfunctional analogue Lys-Asp-Glu-Ala (RTAKDEA). These RTA molecules can enter mammalian cells by fluid phase endocytosis. R TAKDEL was significantly more cytotoxic than either RTA or RTAKDEA to both Vero cells and HeLa cells (250- and 10-fold, respectively), despi te the fact that all these RTA molecules had comparable enzymatic acti vities. This difference did not result from KDEL-mediated binding of R TAKDEL to the cell surface. Enhanced cytotoxicity could be correlated with an increased level of ribosome inactivation, measured as the RTA- catalyzed depurination of 28 S ribosomal RNA. These results indicate t hat the added KDEL sequence facilitated RTA entry into the cytosol. We propose that interaction with the intracellular KDEL receptor promote s retrograde transport of the toxin to the endoplasmic reticulum, wher e translocation of RTA into the cytosol occurs.