Structure And Function Research Of Human AE1(Band 3) C-terminal Domain

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 HCO3-, 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 Ala891-Phe895, Asp896-Glu899, Asp902-Glu906 and Val907-Val911, 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 Ala891-Phe895, Asp896-Glu899, and Val907-Val911 induced high expression of AE1, whereas loss of Asp902-Glu906 results in stable low expression. Pulse chase assay in WT AE1 and its mutants showed that the stability of protein is unaffected by the levels of expression of the AE1 and its mutants. Ala891-Phe895, Asp902-Glu906 and Val907-Val911 mutants exhibited lower levels of trafficking to the plasma membrane compared with WT AE1, while the Asp896-Glu899 mutant was more highly expressed at the plasma membrane. The decreased ability of the mutants to mediate Cl-/HCO3- 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. In addition, the C-terminal 112-residue of band 3 was used to screen the K562 cDNA library. Positive clone plasmids corresponding to p16 and the interaction of p16 with band 3 was confirmed both in yeast and in mammalian cells. Functional experiments show that p16 facilitates the movement of band 3 to plasma membrane with concomitance of increased anion transport activity in 293t cells. Moreover, band 3 up regulates the expression of endogenous p16 in early stage and forced expression of band 3 cooperates with p16 to inhibits the proliferation of HeLa cells. Our findings demonstrate a novel regulation pathway for both band 3 and p16.