Reconstitution of water channel function of aquaporins 1 and 2 by expression in yeast secretory vesicles
Reconstitution of water channel function of aquaporins 1 and 2 by expression in yeast secretory vesicles
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DOI：
10.1152/ajprenal.1998.274.1.f34
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发表时间：
1998-01-01
影响因子：
4.2
通讯作者：
Zeidel, ML
中科院分区：
文献类型：
--
作者：
Coury, LA;Mathai, JC;Zeidel, ML
水通道蛋白 1 (AQP1) 和 2 (AQP2) 在酵母分泌突变体 sec6-4 中表达。该突变体积累高尔基体后质膜靶向囊泡，可用于产生大量膜蛋白。 AQP1 或 AQP2 在酵母中诱导表达，并通过免疫印迹分析定位在分离的 sec6-4 囊泡内。含有 AQP1 和 AQP2 的分泌囊泡表现出高透水性和低水流活化能，表明功能性 AQP1 和 AQP2 的表达。从分泌囊泡中溶解的 AQP1 成功地重构为脂蛋白体，证明了使用酵母系统表达水通道蛋白进行重构研究的能力。与对照囊泡相比，含有 AQP2 的分泌囊泡对甲酰胺、尿素、甘油或质子的渗透性没有增加，这表明 AQP2 对水的选择性高于这些其他物质。我们得出结论，酵母 sec6 囊泡中水通道蛋白的表达是进一步研究哺乳动物水通道功能的有效系统。
Aquaporins 1 (AQP1) and 2 (AQP2) were expressed in the yeast secretory mutant sec6-4. The mutant accumulates post-Golgi, plasma membrane-targeted vesicles and may be used to produce large quantities of membrane proteins. AQP1 or AQP2 were inducibly expressed in yeast and were localized within isolated sec6-4 vesicles by immunoblot analysis. Secretory vesicles containing AQP1 and AQP2 exhibited high water permeabilities and low activation energies for water flow indicating expression of functional AQP1 and AQP2. AQP1 solubilized from secretory vesicles was successfully reconstituted into proteoliposomes, demonstrating the ability to use the yeast system to express aquaporins for reconstitution studies. The AQP2-containing secretory vesicles showed no increased permeability toward formamide, urea, glycerol, or protons compared with control vesicles, demonstrating that AQP2 is highly selective for water over these other substances. We conclude that the expression of aquaporins in yeast sec6 vesicles is a valid system to further study mammalian water channel function.