Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A).
Kidney anion exchanger 1 (kAE1) mediates chloride (Cl⁻) and bicarbonate (HCO₃⁻) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl⁻/HCO₃⁻ exchange at the basolateral membrane and failure of proton (H+) secretion at the apical membrane, causing a kidney disease--distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXØ motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.
Sawasdee
Nunghathai
N
Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Junking
Mutita
M
Ngaojanlar
Piengpaga
P
Sukomon
Nattakan
N
Ungsupravate
Duangporn
D
Limjindaporn
Thawornchai
T
Akkarapatumwong
Varaporn
V
Noisakran
Sansanee
S
Yenchitsomanus
Pa-Thai
PT
D033961:Adaptor Protein Complex 1 / Q000235:genetics / Q000378:metabolism*; D033983:Adaptor Protein Complex mu Subunits / Q000235:genetics / Q000378:metabolism*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C438883:AP1M1 protein, human; D033961:Adaptor Protein Complex 1; D033983:Adaptor Protein Complex mu Subunits; D001457:Anion Exchange Protein 1, Erythrocyte; D001426:Bacterial Proteins; D008164:Luminescent Proteins; C454201:SLC4A1 protein, human; C054562:yellow fluorescent protein, Bacteria