title: Topology studies with biosynthetic fragments identify interacting transmembrane regions of the human red-cell anion exchanger (band 3; AE1).
abstract: The red-cell anion exchanger (band 3; AE1) is a multispanning membrane protein that traverses the bilayer up to 14 times and is N-glycosylated at Asn-642. We have shown that the integrity of six different loops are not essential for stilbene disulphonate-sensitive chloride uptake in Xenopus oocytes. We used an N-glycosylation mutagenesis approach to examine the orientation of the N-terminus and the endogenous glycosylation site of each C-terminal fragment by cell-free translation. The fragments initiating in the loops preceding spans 2, 9 and 11 did not insert into the membrane with the expected orientation. Furthermore, N-glycosylation of Asn-642 might facilitate the membrane integration of span 7. The correct integration of spans 2-3 required the presence of the region containing span 4 and that the luminal exposure of the C-terminus of span 7 is increased in the presence of the region including span 6 or span 8. The results suggest the span 8 region is required for the correct folding of spans 9-10, at least in the presence of the span 11-12 region. Our results suggest that there are intramolecular interactions between the regions of transmembrane spans 1 and 2, 2 and 4, 4 and 5, 7 and 8, 8 and 9-10, and 9-10 and 11-12. Spans 1, 4, 5, 6 and 8 might act as a scaffold for the assembly of spans 2-3, 7 and 9-10. This approach might provide a general method for dissecting the interactions between membrane-spanning regions of polytopic membrane proteins.
authors.affiliation: Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, U.K. J.Groves@tesco.net
authors.forename: J D
authors.initials: JD
authors.lastname: Groves
authors.forename: M J
authors.initials: MJ
authors.lastname: Tanner
mesh_terms: D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry; D017920:Antiporters / Q000737:chemistry*; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides / Q000378:metabolism; D003001:Cloning, Molecular; D004912:Erythrocytes / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D016296:Mutagenesis; D009865:Oocytes; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry; D017510:Protein Folding; D014162:Transfection; D014981:Xenopus
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; D002712:Chlorides; D008565:Membrane Proteins; D010446:Peptide Fragments