Trimming and Readdition of Glucose to N-Linked Oligosaccharides Determines Calnexin Association of a Substrate Glycoprotein in Living Cells*
- From the ‡Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002 and the§Laboratory for Biochemistry, Swiss Federal Institute of Technology, Universitätstrasse 16, CH-8092, Zurich, Switzerland
Abstract
To analyze the role of glucose trimming and reglucosylation in the binding of substrate proteins to calnexin in the endoplasmic reticulum (ER) of living cells, we made use of the thermosensitive vesicular stomatitis virus tsO45 glycoprotein (G protein). At nonpermissive temperature the G protein failed to fold completely and remained bound to calnexin. When the cells were shifted to permissive temperature, complete folding occurred accompanied by glucosidase-mediated elimination of calnexin-G protein complexes. If release from calnexin was blocked during the temperature shift by inhibiting the glucosidases, folding occurred, albeit at a reduced rate. In contrast, when unfolded by a shift from permissive to nonpermissive temperature, the G protein was reglucosylated rapidly and became capable of rebinding to calnexin. The rate at which calnexin binding occurred showed a 20-min delay that was explained by accumulation of the G protein in calnexin-free exit sites of the ER. These contained the glucosyltransferase responsible for reglucosylation of misfolded glycoproteins but had little or no calnexin. After unfolding and reglucosylation, the G proteins moved slowly from these structures back to the ER where they reassociated with the chaperone. Taken together, these results in live cells fully supported the lectin-only model of calnexin function. The ER exit sites emerged as a potentially important location for components of the quality control system.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM 52972, the Mizutani Foundation, and a Bayer scholar award (to K. S. C.) from the Bayer Corporation.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.
We dedicate this paper to the memory of our friend and colleague Dr. Thomas Kreis.
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↵¶ To whom correspondence should be addressed. Fax: 0041-1- 632-1269.
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↵2 K. S. Cannon and A. Helenius, unpublished observations.
- Abbreviations:
- ER
-
endoplasmic reticulum
- GT
-
UDP-glucose:glycoprotein glucosyltransferase
- VSV
-
vesicular stomatitis virus
- G protein
-
glycoprotein
- CHO
-
Chinese hamster ovary
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
- BiP
-
immunoglobulin heavy chain-binding protein
- NB-DNJ
-
N-butyl-1-deoxynojirimycin
- BFA
-
brefeldin A
- Glc1
-
glycan containing a single terminal glucose residue
- Glc0
-
glycan without a terminal glucose residue
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- Received October 9, 1998.
- Revision received December 9, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
