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.
Biochem Biophys Res Commun. 2010 Oct 8;401(1):85-91. doi: 10.1016/j.bbrc.2010.09.015. Epub 2010 Sep 15.
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.
肾脏阴离子交换器 1（kAE1）在肾脏α闰细胞的基底外侧膜上介导氯离子（Cl⁻）和碳酸氢根离子（HCO₃⁻）的交换。kAE1 的转运功能障碍导致基底外侧膜上 Cl⁻/HCO₃⁻交换缺陷和顶端膜上质子（H+）分泌失败，从而导致肾脏疾病-远端肾小管酸中毒（dRTA）。为了更好地了解 kAE1 的转运，我们通过酵母双杂交（Y2H）系统搜索与 kAE1 的 C 末端区域（Ct-kAE1）物理相互作用的蛋白质，该区域包含对细胞内转运至关重要的基序。发现衔接蛋白相关蛋白复合物 1 μ1A（AP-1 mu1A）亚基与 Ct-kAE1 相互作用。通过共免疫沉淀、亲和共纯化、共定位、黄色荧光蛋白（YFP）基于蛋白片段互补测定（PCA）和 GST 下拉测定，在人胚肾（HEK）293T 中证实了 Ct-kAE1 或全长 kAE1 与 AP-1 mu1A 之间的相互作用。发现 AP-1 mu1A 与 Ct-kAE1 的相互作用位点为 Y904DEV907，这是 YXXØ 基序的一个子集。有趣的是，通过小干扰 RNA（siRNA）抑制 HEK 293T 中的内源性 AP-1 mu1A 减少了 kAE1 的膜定位并增加了其细胞内积累，这首次表明 AP-1 mu1A 参与了肾脏α闰细胞的 kAE1 转运。
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