Trimming and readdition of glucose to N-linked oligosaccharides determines calnexin association of a substrate glycoprotein in living cells.

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.

• Authors: K S Cannon, A Helenius
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 274 Issue 11 Pg. 7537-44 (Mar 12 1999) ISSN: 0021-9258 [Print] United States
• PMID: 10066821 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Calcium-Binding Proteins
• Enzyme Inhibitors
• G protein, vesicular stomatitis virus
• Membrane Glycoproteins
• Polysaccharides
• Viral Envelope Proteins
• Calnexin
• Glucosidases
• GTP-Binding Proteins
• Glucose

Topics

• Animals
• CHO Cells
• Calcium-Binding Proteins (metabolism)
• Calnexin
• Cricetinae
• Enzyme Inhibitors (pharmacology)
• GTP-Binding Proteins (metabolism)
• Glucose (metabolism)
• Glucosidases (antagonists & inhibitors)
• Membrane Glycoproteins
• Polysaccharides (metabolism)
• Protein Binding
• Protein Folding
• Viral Envelope Proteins (metabolism)