GLYCOSYLATION OF MULTIPLE EXTRACYTOSOLIC LOOPS IN BAND-3, A MODEL POLYTOPIC MEMBRANE-PROTEIN

N-glycosylated sites in polytopic membrane proteins are usually locali zed to single extracytosolic (EC) loops containing more than 30 residu es [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 transf erase in the lumen of the endoplasmic reticulum. To test this hypothes is, two types of N-glycosylation mutants were constructed using Band 3 , a polytopic membrane protein that contains up to 14 transmembrane se gments and a single endogenous site of N-glycosylation at Asn-642 in E C loop 4. In the first set of mutants, an additional N-glycosylation a cceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutage nesis 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 in to EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed th at microsomes were able to N-glycosylate multiple EC loops in these Ba nd 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. T he localization of N-glycosylation sites to single EC loops in multi-s pan membrane proteins is probably due to the absence of suitably posit ioned acceptor sites on multiple loops.