AE2 anion exchanger polypeptide is a homooligomer in pig gastric membranes: A chemical cross-linking study

Although considerable information is available on the oligomeric states of the AE1 (band 3) anion exchanger, little is known about the physiological s tate of the polypeptides encoded by the nonerythroid AE genes, AE2 and AE3, We have previously characterized the proteolytic susceptibility of native pig gastric AE2. In the course of studies in which pig gastric membranes we re treated with the AE2 transport antagonist, DIDS, we noted evidence for c ross-linking of AE2 proteolytic fragments to higher-order oligomeric forms. We have characterized the ability of DIDS and of selected N-hydroxysuccini mide cross-linking agents to increase the proportion of SDS-resistant oligo mers of pig gastric AE2 and its proteolytic fragments. Cross-linking exhibi ted time and concentration dependence. N-Terminal protein sequencing proved that DIDS treatment created AE2 homodimers, Putative homotetramers were al so observed. Protomers were cross-linked via residues within the C-terminal 40 kDa of AE2. Prior proteolytic cleavage of AE2 in membranes resulted in decreased yield of subsequently cross-linked products. AE2 cross-linking co uld not be detected in membranes pretreated by hypotonic wash and freeze-th aw. The results are interpreted in light of the deduced amino acid sequence of the transmembrane domain of pig AE2.