DeWitt N D, Hong B, Sussman M R, Harper J F
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Plant Physiol. 1996 Oct;112(2):833-44. doi: 10.1104/pp.112.2.833.
More than 11 different P-type H(+)-ATPases have been identified in Arabidopsis by DNA cloning. The subcellular localization for individual members of this proton pump family has not been previously determined. We show by membrane fractionation and immunocytology that a subfamily of immunologically related P-type H(+)-ATPases, including isoforms AHA2 and AHA3, are primarily localized to the plasma membrane. To verify that AHA2 and AHA3 are both targeted to the plasma membrane, we added epitope tags to their C-terminal ends and expressed them in transgenic plants. Both tagged isoforms localized to the plasma membrane, as indicated by aqueous two-phase partitioning and sucrose density gradients. In contrast, a truncated AHA2 (residues 1-193) did not, indicating that the first two transmembrane domains alone are not sufficient for plasma membrane localization. Two epitope tags were evaluated: c-myc, a short, 11-amino acid sequence, and beta-glucuronidase (GUS), a 68-kD protein. The c-myc tag is recommended for its sensitivity and specific immunodetection. GUS worked well as an epitope tag when transgenes were expressed at relatively high levels (e.g. with AHA2-GUS944); however, evidence suggests that GUS activity may be inhibited when a GUS domain is tethered to an H(+)-ATPase complex. Nevertheless, the apparent ability to localize a GUS protein to the plasma membrane indicates that a P-type H(+)-ATPase can be used as a delivery vehicle to target large, soluble proteins to the plasma membrane.
通过DNA克隆，在拟南芥中已鉴定出11种以上不同的P型H(+) - ATP酶。此前尚未确定该质子泵家族各个成员的亚细胞定位。我们通过膜分级分离和免疫细胞化学方法表明，包括异构体AHA2和AHA3在内的免疫相关P型H(+) - ATP酶亚家族主要定位于质膜。为了验证AHA2和AHA3都靶向质膜，我们在它们的C末端添加了表位标签并在转基因植物中进行表达。如双水相分配和蔗糖密度梯度所示，两种带标签的异构体都定位于质膜。相比之下，截短的AHA2（第1 - 193位氨基酸残基）则不然，这表明仅前两个跨膜结构域不足以实现质膜定位。我们评估了两种表位标签：c - myc，一个短的11个氨基酸序列，以及β - 葡萄糖醛酸酶（GUS），一种68kD的蛋白质。推荐使用c - myc标签，因为它具有敏感性和特异性免疫检测能力。当转基因以相对较高水平表达时（例如AHA2 - GUS944），GUS作为表位标签效果良好；然而，有证据表明，当GUS结构域与H(+) - ATP酶复合物相连时，GUS活性可能会受到抑制。尽管如此，将GUS蛋白定位于质膜的明显能力表明，P型H(+) - ATP酶可作为一种递送载体，将大的可溶性蛋白靶向输送到质膜。
