Kissing Sandra, Hermsen Christina, Repnik Urska, Nesset Cecilie Kåsi, von Bargen Kristine, Griffiths Gareth, Ichihara Atsuhiro, Lee Beth S, Schwake Michael, De Brabander Jef, Haas Albert, Saftig Paul
From the Institute of Biochemistry, Christian-Albrechts-University of Kiel, D-24098 Kiel, Germany.
Institute for Cell Biology, Friedrich-Wilhelms University, D-53121 Bonn, Germany.
J Biol Chem. 2015 May 29;290(22):14166-80. doi: 10.1074/jbc.M114.628891. Epub 2015 Apr 22.
The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes.
液泡H⁺-ATP酶（v-ATP酶）复合物对于某些细胞区室的酸化建立和维持起着重要作用，从而确保其功能。最近有人提出，v-ATP酶的跨膜V0区段及其a亚基在胞吞和胞吐途径中促进膜融合，而与其酸化功能无关。在此，我们测试了v-ATP酶这种不依赖质子泵的作用是否也适用于吞噬体-溶酶体融合。令人惊讶的是，缺乏v-ATP酶V0 a3亚基的小鼠胚胎成纤维细胞中的内（溶）酶体正常酸化，并且在有或没有a3亚基的情况下，内体和溶酶体标记蛋白以相似的动力学被募集到吞噬体上。进一步的实验使用了敲低v-ATP酶辅助蛋白2（ATP6AP2）表达的巨噬细胞，导致v-ATP酶V0区段水平大幅降低。然而，酸化似乎未受干扰，并且通过电子显微镜分析，含乳胶珠的吞噬体与溶酶体之间的融合甚至略有增强，V0突变巨噬细胞对非致病性细菌的杀伤也是如此。用药物中和溶酶体pH值并不影响小鼠胚胎细胞或巨噬细胞中吞噬体的成熟。最后，用药物水杨基卤酰胺A将v-ATP酶复合物的两个大的部分锁定在一起，并不抑制体外和细胞内吞噬体与溶酶体的融合。因此，我们的数据并不表明v-ATP酶在吞噬溶酶体形成中具有促进融合的作用。
