Calnexin interaction with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger 1 (band 3).
The interaction of the endoplasmic reticulum chaperone calnexin with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger (AE1), was characterized by cell-free translation and in transfected HEK293 cells, followed by co-immunoprecipitation using anti-calnexin antibody. AE1 contains 12-14 transmembrane segments and has a single site of N-glycosylation at Asn-642 in the fourth extracytosolic loop. This site was mutated (N642D) to create a nonglycosylated protein. Calnexin showed a preferential interaction with N-glycosylated AE1 relative to nonglycosylated AE1 both in vitro and in vivo. This interaction could be blocked by inhibition of glucosidases I and II with castanospermine. Calnexin had access to novel N-glycosylated sites created in other extracytosolic loops in AE1 by site-directed or insertional mutagenesis. The interaction with AE1 was enhanced when multiple sites were introduced into the same loop or into two different loops. An association of calnexin with truncated versions of N-glycosylated AE1 was detected after release of the nascent chains from ribosomes with puromycin. The results show that the interaction of calnexin with the polytopic membrane glycoprotein AE1 was dependent on the presence but not the location of the oligosaccharide. Furthermore, calnexin was associated with AE1 after release of AE1 from the translocation machinery.
Popov
M
M
Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Ontario M5S 1A8, Canada.
Reithmeier
R A
RA
D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism*; D017920:Antiporters / Q000235:genetics / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D002460:Cell Line; D027963:Chloride-Bicarbonate Antiporters; D004912:Erythrocytes / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D014162:Transfection
D001457:Anion Exchange Protein 1, Erythrocyte; D017920:Antiporters; D002135:Calcium-Binding Proteins; D027963:Chloride-Bicarbonate Antiporters; D008562:Membrane Glycoproteins; D037281:Calnexin