Retrograde transport of KDEL-bearing B-fragment of Shiga toxin.
To investigate retrograde transport along the biosynthetic/secretory pathway, we have constructed a recombinant Shiga toxin B-fragment carrying 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 nonfunctional 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 delivered from the plasma membrane, via endosomes and the cisternae of the Golgi apparatus, to the ER. Moreover, the addition of a sulfation site allowed us to show that B-Glyc-KDEL on transit to the ER entered the Golgi apparatus through the trans-Golgi network. Transport of B-Glyc-KDEL to the ER was slowed down by nocodazole, indicating that microtubules are important for the retrograde pathway. Our results document the existence of a continuous pathway from the plasma membrane to the endoplasmic reticulum via the Golgi apparatus and show that a fully folded exogenous protein arriving in the endoplasmic reticulum via this pathway can undergo N-glycosylation.
Johannes
L
L
Institut Curie, CNRS Unité Mixte de Recherche 144, Laboratoire Mécanismes moléculaires du transport intracellulaire, 26 rue d'Ulm, F-75248 Paris Cedex 05, France.
Tenza
D
D
Antony
C
C
Goud
B
B
D001427:Bacterial Toxins / Q000378:metabolism*; D001692:Biological Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015739:Nocodazole / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D021382:Protein Sorting Signals; D022603:Shiga Toxins
D001427:Bacterial Toxins; D009842:Oligopeptides; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D022603:Shiga Toxins; C073762:lysyl-aspartyl-glutamyl-leucine; D015739:Nocodazole