Program in Genetic Models of Disease, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, United States of America.
PLoS One. 2010 Jan 5;5(1):e8585. doi: 10.1371/journal.pone.0008585.
The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.
To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.
We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.
CONCLUSIONS/SIGNIFICANCE: Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.
液泡型 H+-ATP 酶（V-ATPase）是一种高度保守的多亚基酶，它以 ATP 为代价将质子跨膜运输。由此产生的质子梯度具有许多重要功能，其中包括为小分子（如神经递质）的运输提供能量，并使内体等细胞器酸化。该酶不存在于形成吞噬体的质膜中，但通过与已经在膜中带有 V-ATPase 的内体融合而迅速递送至吞噬体。同样，虽然该过程尚未直接可视化，但人们认为该酶在不消化物质的胞吐作用之前从吞噬体膜中被回收。
为了监测 Dictyostelium discoideum 吞噬途径中 V-ATPase 的运输，我们用酵母喂养细胞，酵母是在运输过程中保持形状的大颗粒。为了跟踪 pH 值的变化，我们用荧光素异硫氰酸酯将酵母偶联。细胞与 VatM-GFP 一起标记，VatM-GFP 是 V-ATPase 的荧光标记跨膜亚基，同时与阶段特异性内体标记物或与 mRFP 标记的细胞骨架蛋白组合标记。
我们发现，V-ATPase 在胞吐作用前不久通过小泡化从吞噬体膜中通常被回收。然而，如果细胞被限制在小空间中，大的吞噬体可能会过早地被胞吐。在这种情况下，一个富含 V-ATPase 的大泡状体通常在胞吐作用前不久从酸性吞噬体中分离出来。这个泡状体由肌动蛋白尾巴推动，很快获得早期内体的特性，揭示了 V-ATPase 快速回收的意外机制。任何到达质膜的 V-ATPase 也会被迅速回收。
结论/意义：因此，活细胞显微镜揭示了 V-ATPase 在胞吞途径中回收的常见途径和替代途径。
