Department of Biochemistry, Biotechnology Center, University of Wisconsin , 425 Henry Mall, Madison, Wisconsin 53706, United States.
Biochemistry. 2014 Jan 28;53(3):566-78. doi: 10.1021/bi401096m. Epub 2014 Jan 15.
The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an 11 gene family and are collectively essential for embryo development. We report the translational fusion of a tandem affinity-purification tag to the 5' end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryonic lethal phenotype of endogenous double aha1/aha2 knockdowns. Western blots of SDS-PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions and indicate a hexameric quaternary structure. TAP-tagged AHA1 rescue lines exhibited reduced vertical root growth. Analysis of the plasma membrane and soluble proteomes identified several plasma membrane-localized proteins with alterred abundance in TAP-tagged AHA1 rescue lines compared to wild type. Using affinity-purification mass spectrometry, we uniquely identified two additional AHA isoforms, AHA9 and AHA11, which copurified with TAP-tagged AHA1. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP-tagged AHA1 transgene has complemented all essential endogenous AHA1 and AHA2 functions and have shown that these plants can be used to purify AHA1 protein and to identify in planta interacting proteins by mass spectrometry.
拟南芥质膜质子 ATP 酶基因 AHA1 和 AHA2 是 11 个基因家族中表达水平最高的两个亚型，对于胚胎发育是必需的。我们报告了在一个基因组克隆中，AHA1 开放阅读框 5' 端串联亲和纯化标签的翻译融合。在拟南芥中稳定表达 TAP 标记的 AHA1 可挽救内源性双 aha1/aha2 敲低的胚胎致死表型。SDS-PAGE 和 Blue Native 凝胶的 Western blot 显示 AHA1 在质膜部分富集，并表明其具有六聚体四级结构。TAP 标记的 AHA1 挽救系表现出垂直根生长减少。质膜和可溶性蛋白质组分析鉴定了几种质膜定位蛋白，与 TAP 标记的 AHA1 挽救系相比，其丰度发生改变。使用亲和纯化质谱分析，我们独特地鉴定了另外两种 AHA 亚型 AHA9 和 AHA11，它们与 TAP 标记的 AHA1 共纯化。总之，我们生成了转基因拟南芥系，其中 TAP 标记的 AHA1 转基因可补充所有必需的内源性 AHA1 和 AHA2 功能，并且表明这些植物可用于通过质谱法纯化 AHA1 蛋白和鉴定植物体内相互作用蛋白。
