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Abstract：
Regulation of the luminal pH of late endocytic compartments in continuously fed mammalian cells is poorly understood. Using normal rat kidney fibroblasts, we investigated the reversible assembly/disassembly of the proton pumping V-ATPase when endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. We took advantage of previous work showing that sucrosomes formed by the uptake of sucrose are swollen endolysosomes from which lysosomes are reformed after uptake of invertase. Using confocal microscopy and subcellular fractionation of NRK cells stably expressing fluorescently tagged proteins, we found net recruitment of the V1 subcomplex during sucrosome formation and loss during lysosome reformation, with a similar time course to RAB7a loss. Addition of invertase did not alter mTORC1 signalling, suggesting that the regulation of reversible V-ATPase assembly/disassembly in continuously fed cells differs from that in cells subject to amino acid depletion/refeeding. Using live cell microscopy, we demonstrated recruitment of a fluorescently tagged V1 subunit during endolysosome formation and a dynamic equilibrium and rapid exchange between the cytosolic and membrane bound pools of this subunit. We conclude that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells.
摘要：
对连续喂食的哺乳动物细胞中晚期胞吞区室的腔pH的调节知之甚少。使用正常大鼠肾成纤维细胞，我们研究了通过接吻和融合晚期内体与溶酶体形成内溶酶体以及随后的溶酶体重组过程中，质子泵V-ATPase的可逆组装/分解。我们利用先前的工作表明，通过吸收蔗糖而形成的蔗糖体是溶胀的内溶酶体，在吸收转化酶后，溶酶体会从该溶酶体中重新形成。使用共聚焦显微镜和稳定表达荧光标记蛋白的NRK细胞的亚细胞分级分离，我们发现在蔗糖体形成过程中V1亚复合物的净募集和溶酶体重整过程中的损失，与RAB7a损失的时间过程相似。添加转化酶不会改变mTORC1信号，表明连续喂食的细胞中可逆的V-ATPase组装/分解的调节不同于氨基酸消耗/重新喂食的细胞。使用活细胞显微镜，我们证明了在内溶酶体形成过程中荧光标记的V1亚基的募集以及该亚基的胞浆和膜结合池之间的动态平衡和快速交换。我们得出的结论是，可逆的V-ATPase组装/拆解在连续喂食的细胞中调节内溶酶体/溶酶体pH中起着关键作用。[媒体:见文本][媒体:见文本][媒体:见文本][媒体:见文本]。
