Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin*
- From the Institut Curie, CNRS Unité Mixte de Recherche 144, Laboratoire Mécanismes moléculaires du transport intracellulaire, 26 rue d’Ulm, F-75248 Paris Cedex 05, France
Abstract
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.
Footnotes
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↵* This work was supported in part by Association de la Recherche contre le Cancer Grant 1405, by the Fondation pour la Recherche Médicale, by Human Frontier Science Program RG 432/96 Grant, and by European Union Grant ERB CHR XCT 940592.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Supported by a grant from the BASF-Forschungsprogramm/Studienstiftung des deutschen Volkes.
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↵§ To whom correspondence should be addressed: Institut Curie, CNRS UMR 144, 26 rue d’Ulm, F-75248 Paris Cedex 05, France. Tel.: 33-1-42-34-63-81; Fax: 33-1-42-34-63-82; E-mail: bgoud{at}curie.fr.
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↵1 The abbreviations used are: ER, endoplasmic reticulum; TGN, trans-Golgi network; Lamp, lysosomal-associated protein; PBS, phosphate-buffered saline; DMM, 1-deoxymannojirimicin; DTAF, 5([4,6-dichlorotriazin-2-yl]amino)fluorescein.
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↵2 L. Johannes and B. Goud, unpublished observations.
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- Received February 10, 1997.
- Revision received May 9, 1997.
