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首页> 外文期刊>The FEBS journal >The proximity between C-termini of dimeric vacuolar H-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique
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The proximity between C-termini of dimeric vacuolar H-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique

机译：使用原子力显微镜和金纳米粒子技术测定的二聚液泡H-焦磷酸酶C末端之间的接近性

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摘要

Vacuolar H-translocating inorganic pyrophosphatase [vacuolar H-pyrophosphatase (V-PPase); EC 3.6.1.1] is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen, at the expense of PPi hydrolysis, for the storage of ions, sugars, and other metabolites. Dimerization of V-PPase is necessary for full proton translocation function, although the structural details of V-PPase within the vacuolar membrane remain uncertain. The C-terminus presumably plays a crucial role in sustaining enzymatic and proton-translocating reactions. We used atomic force microscopy to visualize V-PPases embedded in an artificial lipid bilayer under physiological conditions. V-PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V-PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V-PPase protrusions were 2.8 pl 0.3 nm and 26.3 pl 4.7 nm, whereas those of the luminal protrusions were 1.2 pl 0.1 nm and 21.7 pl 3.6 nm, respectively. Moreover, both C-termini of dimeric subunits of V-PPase are on the same side of the membrane, and they are close to each other, as visualized with antibody and gold nanoparticles against 6xHis tags on C-terminal ends of the enzyme. The distance between the V-PPase C-terminal ends was determined to be approximately 2.2 pl 1.4 nm. Thus, our study is the first to provide structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini.
机译：液泡H易位的无机焦磷酸酶[液泡H-焦磷酸酶（V-PPase）; EC 3.6.1.1]是同型二聚体质子转位酶;它在质子从细胞质到液泡腔的电迁移中起着关键作用，但以PPi水解为代价，用于离子，糖和其他代谢物的存储。 V-PPase的二聚化对于完整的质子转运功能是必需的，尽管液泡膜中V-PPase的结构细节仍不确定。 C端大概在维持酶促和质子移位反应中起关键作用。我们使用原子力显微镜在生理条件下可视化嵌入人工脂质双层中的V-PPase。 V-PPases随机分布在重组脂质双层中。约43.3％的V-PPase突出物面对细胞质，而56.7％的细胞面对液泡腔。胞质V-PPase突起的平均高度和宽度分别为2.8 pl 0.3 nm和26.3 pl 4.7 nm，而管腔突起的平均高度和宽度分别为1.2 pl 0.1 nm和21.7 pl 3.6 nm。此外，V-PPase的二聚体亚基的两个C末端都在膜的同一侧，并且彼此靠近，如用抗体和针对该酶C末端的6xHis标签的金纳米颗粒所观察到的那样。 V-PPase C末端之间的距离被确定为大约2.2μl1.4nm。因此，我们的研究首次提供了膜结合的V-PPase二聚体的结构细节，揭示了其邻近的C-末端。

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