Jouret François, Courtoy Pierre J, Devuyst Olivier
Division of Nephrology, Université Catholique de Louvain Medical School, B-1200, Brussels, Belgium.
Methods Mol Biol. 2011;741:285-99. doi: 10.1007/978-1-61779-117-8_19.
Besides its location at the plasma membrane, CFTR is present in intracellular vesicles along both the exocytic and the endocytic pathways. Immunostaining and subcellular fractionation studies of mouse kidney demonstrate that CFTR is located in endosomes of the cells lining the terminal part of the proximal tubule (PT). The PT cells efficiently reabsorb the ultrafiltered low molecular weight (LMW) proteins by apical endocytosis involving the multiligand receptors megalin and cubilin. The progression from early endosomes to lysosomes depends on the integrity of the cytoskeleton, as well as on vesicular acidification. The latter is mediated by the vacuolar H+-ATPase (V-ATPase) and requires an anionic conductance to dissipate the transmembrane potential gradient. CFTR might ensure such chloride conductance, thereby participating to endosomal acidification and protein uptake by PT cells. Immunostaining with well-characterized antibodies shows that CFTR is located in the terminal segment of PT, where it co-distributes with megalin and cubilin. Subcellular fractionation of total mouse kidneys through Percoll gradients demonstrates the co-localization of CFTR with the V-ATPase and early endosome markers including the Cl-/H+ exchanger, ClC-5, and the small GTPase, Rab5a. Deglycosylation studies and immunoblotting show a distinct glycosylation pattern for CFTR in mouse kidney and lung. The segmental and subcellular distribution of CFTR in mouse kidney supports a role for CFTR in PT receptor-mediated endocytosis of ultrafiltered LMW proteins.
除了位于质膜外，囊性纤维化跨膜传导调节因子（CFTR）还存在于沿胞吐和胞吞途径的细胞内囊泡中。对小鼠肾脏的免疫染色和亚细胞分级分离研究表明，CFTR位于近端小管（PT）末端部分细胞的内体中。PT细胞通过涉及多配体受体巨膜蛋白和立方蛋白的顶端胞吞作用有效地重吸收超滤后的低分子量（LMW）蛋白质。从早期内体到溶酶体的进程取决于细胞骨架的完整性以及囊泡酸化。后者由液泡H⁺-ATP酶（V-ATP酶）介导，并且需要阴离子传导来消散跨膜电位梯度。CFTR可能确保这种氯离子传导，从而参与PT细胞的内体酸化和蛋白质摄取。用特性明确的抗体进行免疫染色表明，CFTR位于PT的末端节段，与巨膜蛋白和立方蛋白共同分布。通过Percoll梯度对整个小鼠肾脏进行亚细胞分级分离，证明CFTR与V-ATP酶以及早期内体标记物共定位，这些标记物包括Cl⁻/H⁺交换体ClC-5和小GTP酶Rab5a。去糖基化研究和免疫印迹显示，CFTR在小鼠肾脏和肺中有明显不同的糖基化模式。CFTR在小鼠肾脏中的节段性和亚细胞分布支持其在PT受体介导的超滤LMW蛋白质胞吞作用中的作用。
