N-glycosylated sites in polytopic membrane proteins are usually localized to single extracytosolic (EC) loops containing more than 30 residues [Landolt-Marticorena and Reithmeier (1994) Biochem. J. 302, 253–260]. This may be due to a biosynthetic restriction whereby only a single loop of nascent polypeptide is available to the oligosaccharyl transferase in the lumen of the endoplasmic reticulum. To test this hypothesis, two types of N-glycosylation mutants were constructed using Band 3, a polytopic membrane protein that contains up to 14 transmembrane segments and a single endogenous site of N-glycosylation at Asn-642 in EC loop 4. In the first set of mutants, an additional N-glycosylation acceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutagenesis in EC loop 3, with or without retention of the endogenous site. In the second set of mutants, EC loop 4 was duplicated and inserted into EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed that microsomes were able to N-glycosylate multiple EC loops in these Band 3 mutants. The acceptor site in EC loop 3 was poorly N-glycosylated, probably due to the suboptimal size (25 residues) of this EC loop. The localization of N-glycosylation sites to single EC loops in multi-span membrane proteins is probably due to the absence of suitably positioned acceptor sites on multiple loops.
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September 1996
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Research Article|
September 01 1996
Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein
Lisa Y TAM;
Lisa Y TAM
1MRC Group in Membrane Biology, Departments of Medicine and Biochemistry, Room 7344, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada, M5S 1A8
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Carolina LANDOLT-MARTICORENA;
Carolina LANDOLT-MARTICORENA
1MRC Group in Membrane Biology, Departments of Medicine and Biochemistry, Room 7344, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada, M5S 1A8
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Reinhart A. F. REITHMEIER
Reinhart A. F. REITHMEIER
*
1MRC Group in Membrane Biology, Departments of Medicine and Biochemistry, Room 7344, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada, M5S 1A8
*To whom correspondence should be addressed.
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Biochem J (1996) 318 (2): 645–648.
Article history
Received:
March 15 1996
Revision Received:
May 03 1996
Accepted:
May 08 1996
Citation
Lisa Y TAM, Carolina LANDOLT-MARTICORENA, Reinhart A. F. REITHMEIER; Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein. Biochem J 1 September 1996; 318 (2): 645–648. doi: https://doi.org/10.1042/bj3180645
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