MAPPING THE ENDS OF TRANSMEMBRANE SEGMENTS IN A POLYTOPIC MEMBRANE-PROTEIN - SCANNING N-GLYCOSYLATION MUTAGENESIS OF EXTRACYTOSOLIC LOOPS IN THE ANION-EXCHANGER, BAND-3

Band 3, the anion exchanger of human erythrocytes, contains up to 14 t ransmembrane (TM) segments and has a single endogenous site of N-glyco sylation at Asn(642) in extracellular (EC) loop 4, The requirements fo r N-glycosylation of EC loops and the topology of this :polytopic memb rane protein were determined by scanning N-glycosylation mutagenesis a nd cell-free translation in a reticulocyte lysate supplemented with mi crosomal membranes. The endogenous and novel acceptor sites located ne ar the middle of the 35 residue EC loop 4 were efficiently N-glycosyla ted; however, no N-glycosylation occurred at sites located within shar ply defined regions close to the adjacent TM segments, Acceptor sites located in the center of EC loop 3, which contains 25 residues, were p oorly N-glycosylated, Expansion of this loop with a 4-residue insert c ontaining an acceptor site increased N-glycosylation, Acceptor sites l ocated in short (<10 residues) loops (putative EC loops 1, 2, 6, and 7 ) were not N-glycosylated; however, insertion of EC loop 4 into EC loo ps 1, 2, or 7, but not 6, resulted in efficient N-glycosylation, Accep tor sites in putative intracellular (IC) loop 5 exhibited a similar pa ttern of N-glycosylation as EC loop 4, indicating a lumenal dispositio n during biosynthesis, To be efficiently N-glycosylated, EC loops in p olytopic membrane proteins must be larger than 25 residues in size, wi th acceptor sites located greater than 12 residues away from the prece ding TM segment and greater than 14 residues away from the following T M segment, Application of this requirement allowed a significant refin ement of the topology of Band 3 including a more accurate mapping of t he ends of TM segments, The strict distance dependence for N-glycosyla tion of loops suggests that TM segments in polytopic membrane proteins are held quite precisely within the translocation machinery during th e N-glycosylation process.