Kanadaptin (kidney
anion exchanger adaptor
protein) has recently been identified as a
protein with binding activity to the cytoplasmic domain of the kidney Na(+)-independent Cl(-)/HCO(-)(3)
anion exchanger 1 (kAE1). Since it is widely expressed in tissues devoid of kAE1, however, kanadaptin is likely to have additional cellular roles. This is supported by its multidomain structure, and possession of three clusters of
basic amino acids exhibiting similarity to known nuclear localization sequences (NLSs). In the present study, we use immunofluorescence and subcellular fractionation approaches to demonstrate that kanadaptin is localized within the nuclei of various epithelial and non-epithelial cultured cell types. The role of the different NLSs is examined in transfection studies using plasmids encoding full-length kanadaptin with or without
green fluorescent protein (GFP) as a fusion tag, as well as truncation derivatives thereof. Strong nuclear localization of fusion
proteins containing
amino acids 140-230 of kanadaptin, which include the sequence AVSRKRKA(193) (NLS1) was observed. Substitution of Arg(191) with a
threonine residue resulted in a cytoplasmic location of the expressed
protein, while NLS1 proved sufficient to target an otherwise cytoplasmically localized
beta-galactosidase-GFP fusion
protein to the nucleus. Using a direct binding assay we show that a fusion
protein containing kanadaptin
amino acids 1-231 (but not the Thr(191) substituted derivative) is recognized with nM affinity by the conventional NLS-binding
importin alpha/beta heterodimer. Nuclear import studies on microinjected and permeabilized rat
hepatoma cells demonstrated functionality of the NLS in nuclear targeting, with inhibition by
antibodies demonstrating the requirement of both
importin alpha and beta for nuclear import of kanadaptin. That kanadaptin possesses a functional
importin-alpha/beta-recognized NLS explains the nuclear localization of kanadaptin in various cultured cell types, and opens up the possibility that kanadaptin may have a signalling role in the nucleus.