Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK

Michelle E. Maxson, Y. M. Abbas, J. Wu, S. Grinstein, J. Rubinstein
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引用次数: 10

Abstract

Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive nor specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases in eukaryotic cells. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with AlexaFluor-568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on the subcellular localization of the lysosome.
利用军团菌效应蛋白SidK检测和定量液泡H+ atp酶
分泌和内吞细胞器的酸化是适当的受体循环、膜运输、蛋白质降解和溶质运输所必需的。质子泵送液泡atp酶(v - atp酶)负责这种腔内酸化,随着分泌和内吞囊泡的成熟,这种酸化逐渐增加。v - atp酶复合物的密度增加被认为是pH逐渐下降的原因,但现有的试剂没有足够的灵敏度和特异性来测试这一假设。我们介绍了一种新的探针来定位和量化真核细胞中的v - atp酶。该探针来源于SidK,一种与v - atp酶a亚基结合的嗜肺军团菌效应蛋白。我们制备了编码SidK1-278荧光嵌合体的质粒,并用AlexaFluor-568标记重组SidK1-278,分别在活细胞和固定细胞中以高特异性可视化和定量V-ATPases。我们发现v - atp酶是在吞噬体成熟过程中逐渐获得的,它们分布在离散的膜亚结构域中,它们在溶酶体中的密度取决于溶酶体的亚细胞定位。
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