Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein.
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.
Tam
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Department of Medicine, University of Toronto, Ontario, Canada.
Landolt-Marticorena
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Reithmeier
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D000595:Amino Acid Sequence; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000096:biosynthesis / Q000737:chemistry*; D001216:Asparagine; D002474:Cell-Free System; D006031:Glycosylation; D006801:Humans; D008861:Microsomes / Q000378:metabolism; D008961:Models, Structural; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic
D001457:Anion Exchange Protein 1, Erythrocyte; D011994:Recombinant Proteins; D001216:Asparagine