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Abstract
Kanadaptin (k¯idney anion exchanger adaptor protein) is a widely expressed protein, shown previously to interact with the cytosolic domain of mouse Cl−/HCO3− anion exchanger 1 (kAE1) but not erythroid AE1 (eAE1) by a yeast-two hybrid assay. Kanadaptin was co-localized with kAE1 in intracellular membranes but not at the plasma membrane in α-intercalated cells of rabbit kidney. It was suggested that kanadaptin is an adaptor protein or chaperone involved in targeting kAE1 to the plasma membrane. To test this hypothesis, the interaction of human kanadaptin with human kAE1 was studied in co-transfected HEK293 cells. Human kanadaptin contains 796 amino acids and was immuno-detected as a 90 kDa protein in transfected cells. Pulse-chase experiments showed that it has a half-life (t1/2) of 7 h. Human kanadaptin was localized predominantly to the nucleus, whereas kAE1 was present intracellularly and at the plasma membrane. Trafficking of kAE1 from its site of synthesis in the endoplasmic reticulum to the plasma membrane was unaffected by co-expression of human kanadaptin. Moreover, we found that no interaction between human kanadaptin and kAE1 or eAE1 could be detected in co-transfected cells either by co-immunoprecipitation or by histidine6-tagged co-purification. Taken together, we found that human kanadaptin did not interact with kAE1 and had no effect on trafficking of kAE1 to the plasma membrane in transfected cells. Kanadaptin may not be involved in the biosynthesis and targeting of kAE1. As such, defects in kanadaptin and its interaction with kAE1 are unlikely to be involved in the pathogenesis of the inherited kidney disease, distal renal tubular acidosis (dRTA).
dRTA, distal renal tubular acidosis; C12E8, octaethylene glyco mono-n-dodecyl-ether; eAE1, erythroid anion exchanger1; endo H, endoglycosidase H; GPA, glycophorin A; HEK, human embryonic kidney; kAE1, kidney anion exchanger 1; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PCR-SSCP, polymerase chain reaction-single stranded conformation polymorphism; PMSF, phenylmethylsulfonyl fluoride; PNGase F, peptide N-glycanase F; SDS, sodium dodecyl sulfate
dRTA, distal renal tubular acidosis; C12E8, octaethylene glyco mono-n-dodecyl-ether; eAE1, erythroid anion exchanger1; endo H, endoglycosidase H; GPA, glycophorin A; HEK, human embryonic kidney; kAE1, kidney anion exchanger 1; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PCR-SSCP, polymerase chain reaction-single stranded conformation polymorphism; PMSF, phenylmethylsulfonyl fluoride; PNGase F, peptide N-glycanase F; SDS, sodium dodecyl sulfate