Vacuolar ATPase in Phagosome-Lysosome Fusion
Vacuolar ATPase in Phagosome-Lysosome Fusion
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DOI：
10.1074/jbc.m114.628891
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发表时间：
2015-05-29
影响因子：
4.8
通讯作者：
Saftig, Paul
中科院分区：
文献类型：
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作者：
Kissing, Sandra;Hermsen, Christina;Saftig, Paul
液泡 H+ -ATP 酶 (v-ATP 酶) 复合物有助于建立和维持某些细胞区室的酸化，从而确保其功能。最近有人提出，v-ATP酶的跨膜V-0区段及其a-亚基促进内吞和胞吐途径中的膜融合，与其酸化功能无关。在这里，我们测试了 v-ATP 酶的这种质子泵独立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 V-0 a3 亚基的小鼠胚胎成纤维细胞中的内（溶酶）体正常酸化，并且在存在或不存在 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招募到吞噬体中。进一步的实验使用了 v-ATPase 辅助蛋白 2 (ATP6AP2) 表达敲低的巨噬细胞，导致 v-ATPase V-0 部分的水平大大降低。然而，酸化似乎未受干扰，并且通过电子显微镜分析，含有乳胶珠的吞噬体和溶酶体之间的融合甚至略有增强，V-0突变型巨噬细胞对非病原细菌的杀伤力也是如此。药理学中和的溶酶体 pH 值不会影响小鼠胚胎细胞或巨噬细胞中吞噬体的成熟。最后，通过药物水利苯卤酰胺 A 将 v-ATP 酶复合物的两大部分锁定在一起，在体外和在吞噬体与溶酶体的细胞融合中不会抑制。因此，我们的数据并不表明 v-ATP 酶在吞噬溶酶体的形成中具有促进融合的作用。
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 V-0 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 V-0 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 V-0 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 V-0 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.