title: Effect of block deletions in the C-terminus on the functional expression of human anion exchanger 1 (AE1).
abstract: The human anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is responsible for the exchange of Cl(-) for HCO(3)(-). However, the detailed role played by the AE1 C-terminal region in the anion translocation and membrane trafficking process remains unclear. In this paper, we created four mutants in the human AE1 C-terminus by deletion of the residues Ala(891)-Phe(895), Asp(896)-Glu(899), Asp(902)-Glu(906) and Val(907)-Val(911), to investigate the role of these sequences in functional expression of AE1. WT AE1 and its deletion mutant constructs were expressed in HEK 293 cells. Western blotting showed that deletions of Ala(891)-Phe(895), Asp(896)-Glu(899), and Val(907)-Val(911) induced high expression of AE1, whereas loss of Asp(902)-Glu(906) results in stable low expression. Pulse chase assays of WT AE1 and its mutants showed that the stability of protein is unaffected by the levels of expression of the AE1 and its mutants. Ala(891)-Phe(895), Asp(902)-Glu(906) and Val(907)-Val(911) mutants exhibited lower levels of trafficking to the plasma membrane compared with WT AE1, while the Asp(896)-Glu(899) mutant was more highly expressed at the plasma membrane. The decreased ability of the mutants to mediate Cl(-)/HCO(3)(-) exchange in transfected cells revealed that the deletion sequences have an important role in transport activity. These results demonstrate that the studied residues in the AE1 C-terminus differently affect the expression, membrane trafficking and functional folding of AE1.
authors: Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Rui-Jin Hospital, Shanghai Jiao-Tong University, School of Medicine/Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, PR China.
authors: Yong
authors: Y
authors: Wang
authors: Shao-Fang
authors: SF
authors: Wu
authors: Guo-Qiang
authors: GQ
authors: Chen
authors: Guo-Hui
authors: GH
authors: Fu
mesh_terms: D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000378:metabolism*; D001224:Aspartic Acid; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D021381:Protein Transport; D017384:Sequence Deletion / Q000235:genetics*; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D050505:Mutant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid