Topology of transmembrane segments 1-4 in the human chloride/bicarbonate anion exchanger 1 (AE1) by scanning N-glycosylation mutagenesis.
Human AE1 (anion exchanger 1), or Band 3, is an abundant membrane glycoprotein found in the plasma membrane of erythrocytes. The physiological role of the protein is to carry out chloride/bicarbonate exchange across the plasma membrane, a process that increases the carbon-dioxide-carrying capacity of blood. To study the topology of TMs (transmembrane segments) 1-4, a series of scanning N-glycosylation mutants were created spanning the region from EC (extracellular loop) 1 to EC2 in full-length AE1. These constructs were expressed in HEK-293 (human embryonic kidney) cells, and their N-glycosylation efficiencies were determined. Unexpectedly, positions within putative TMs 2 and 3 could be efficiently glycosylated. In contrast, the same positions were very poorly glycosylated when present in mutant AE1 with the SAO (Southeast Asian ovalocytosis) deletion (DeltaA400-A408) in TM1. These results suggest that the TM2-3 region of AE1 may become transiently exposed to the endoplasmic reticulum lumen during biosynthesis, and that there is a competition between proper folding of the region into the membrane and N-glycosylation at introduced sites. The SAO deletion disrupts the proper integration of TMs 1-2, probably leaving the region exposed to the cytosol. As a result, engineered N-glycosylation acceptor sites in TM2-3 could not be utilized by the oligosaccharyltransferase in this mutant form of AE1. The properties of TM2-3 suggest that these segments form a re-entrant loop in human AE1.
Cheung
Joanne C
JC
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8.
Li
Jing
J
Reithmeier
Reinhart A F
RA
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry*; D001665:Binding Sites; D002460:Cell Line; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding
D001457:Anion Exchange Protein 1, Erythrocyte