Johannes L, Tenza D, Antony C, Goud B
Institut Curie, CNRS Unité Mixte de Recherche 144, Laboratoire Mécanismes moléculaires du transport intracellulaire, 26 rue d'Ulm, F-75248 Paris Cedex 05, France.
J Biol Chem. 1997 Aug 1;272(31):19554-61. doi: 10.1074/jbc.272.31.19554.
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.
为了研究沿生物合成/分泌途径的逆向转运，我们构建了一种重组志贺毒素B片段，其羧基末端带有一个N-糖基化位点和一个KDEL回收基序（B-Glyc-KDEL）。与HeLa细胞孵育后，B-Glyc-KDEL在内质网（ER）中逐渐被糖基化，并与该区室稳定结合。具有无功能KDEL序列的B片段（B-Glyc-KDELGL）的糖基化动力学与B-Glyc-KDEL大致相同，但在稳态时定位于高尔基体。形态学研究表明，B-Glyc-KDEL从质膜通过内体和高尔基体的潴泡被转运到内质网。此外，添加一个硫酸化位点使我们能够证明，转运到内质网的B-Glyc-KDEL通过反式高尔基体网络进入高尔基体。诺考达唑减缓了B-Glyc-KDEL向内质网的转运，表明微管对逆向途径很重要。我们的结果证明了从质膜经高尔基体到内质网存在一条连续途径，并表明通过该途径到达内质网的完全折叠的外源蛋白可以进行N-糖基化。
