RETROGRADE TRANSPORT OF KDEL-BEARING B-FRAGMENT OF SHIGA TOXIN

To investigate retrograde transport along the biosynthetic/secretory p athway, we have constructed a recombinant Shiga toxin B-fragment carry ing an N-glycosylation site and a KDEL retrieval motif at its carboxyl terminus (B-Glyc-KDEL), After incubation with HeLa cells, B-Glyc-KDEL was progressively glycosylated in the endoplasmic reticulum (ER) and remained stably associated with this compartment. B-fragment with a no nfunctional KDEL sequence (B-Glyc-KDELGL) was glycosylated with about the same kinetics as B-Glyc-KDEL but localized at steady state to the Golgi apparatus. Morphological studies showed that B-Glyc-KDEL was del ivered from the plasma membrane, via endosomes and the cisternae of th e Golgi apparatus, to the ER. Moreover, the addition of a sulfation si te allowed us to show that B-Glyc-KDEL on transit to the ER entered th e Golgi apparatus through the trans-Golgi network. Transport of B-Glyc -KDEL to the ER was slowed down by nocodazole, indicating that microtu bules are important for the retrograde pathway. Our results document t he existence of a continuous pathway from the plasma membrane to the e ndoplasmic reticulum via the Golgi apparatus and show that a fully fol ded exogenous protein arriving in the endoplasmic reticulum via this p athway can undergo N-glycosylation.