Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes
Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes
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DOI：
10.1074/jbc.m011577200
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发表时间：
2001-05-25
影响因子：
4.8
通讯作者：
Marshansky, V
中科院分区：
文献类型：
--
作者：
Maranda, B;Brown, D;Marshansky, V
肾近端小管上皮细胞具有广泛的顶端内吞装置，其对于穿过肾小球滤过屏障的蛋白质的重吸收和降解至关重要，并且还参与功能重要的顶端质膜转运蛋白的广泛再循环。我们在这里表明，Arf-核苷酸交换因子，ARNO（ADP-核糖基化因子核苷酸位点开放器）以及Arf 6和Arf 1小GTP酶位于肾近端小管受体介导的内吞途径，和ARNO和Arf 6招聘从胞质到内涵体是pH值依赖性的。在近曲小管中，ARNO和Arf 6与V-ATP酶共定位于近曲小管顶端的内体，Arf 1定位于近曲小管上皮细胞的顶端，也定位于高尔基体。Western blot分析表明，ARNO、Arf 6和Arf 1在纯化的内体和近曲小管胞质中均有表达，一个易位分析表明，ATP驱动的内体酸化触发招聘ARNO和Arf 6从近端小管胞质溶胶到内体膜。ARNO和Arf 6的易位被逆转的V型ATP酶抑制剂和解偶联剂的内体腔内pH值，并与内体酸化的幅度。我们的数据表明，V型ATP酶依赖的酸化刺激ARNO和Arf 6选择性募集到近端小管早期内体，这一机制可能在近端小管受体介导的内吞作用的原位pa依赖性调节中起重要作用。
Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules, Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi, By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol, A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes, This mechanism may play an important role in the pa-dependent regulation of receptor-mediated endocytosis in proximal tubules in situ.