Junking Mutita, Sawasdee Nunghathai, Duangtum Natapol, Cheunsuchon Boonyarit, Limjindaporn Thawornchai, Yenchitsomanus Pa-thai
Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Traffic. 2014 Jul;15(7):788-802. doi: 10.1111/tra.12172. Epub 2014 May 5.
Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells.
肾阴离子交换蛋白1（kAE1）通过在远端肾单位α-闰细胞基底外侧膜介导氯/碳酸氢根（Cl-/HCO3-）交换，在酸碱平衡中发挥重要作用。由编码kAE1的SLC4A1突变导致的kAE1细胞内运输受损会引发肾脏疾病——远端肾小管酸中毒（dRTA）。然而，目前尚不清楚kAE1从反式高尔基体网络（TGN）到基底外侧膜的细胞内分选和运输过程是如何发生的。在此，我们研究了基底外侧相关分选蛋白，包括衔接蛋白（AP）复合物的μ1亚基、网格蛋白和蛋白激酶D，在极化和非极化肾细胞中对kAE1运输的作用。通过使用RNA干扰、免疫共沉淀、基于黄色荧光蛋白的蛋白片段互补分析和免疫荧光染色，我们证明了AP-1 μ1A、AP-3 μ1、AP-4 μ1和网格蛋白（而非AP-1 μ1B、PKD1或PKD2）在kAE1的细胞内分选和运输中起关键作用。我们还通过双重免疫荧光染色证明了kAE1与基底外侧相关分选蛋白在人肾组织中的共定位。这些发现表明，AP-1 μ1A、AP-3 μ1、AP-4 μ1和网格蛋白是kAE1从TGN分选和运输到分泌酸的α-闰细胞基底外侧膜所必需的。
