Yang Haibing, Murphy Angus S
Department of Horticulture, Purdue University, West Lafayette, IN 47907-2010, USA.
Plant J. 2009 Jul;59(1):179-91. doi: 10.1111/j.1365-313X.2009.03856.x. Epub 2009 Feb 27.
Heterologous expression systems based on tobacco BY-2 cells, Arabidopsis cell cultures, Xenopus oocytes, Saccharomyces cerevisiae, and human HeLa cells have been used to express and characterize PIN, ABCB (PGP), and AUX/LAX auxin transporters from Arabidopsis. However, no single system has been identified that can be used for effective comparative analyses of these proteins. We have developed an accessible Schizosaccharomyces pombe system for comparative studies of plant transport proteins. The system includes knockout mutants in all ABC and putative auxin transport genes and Gateway((R))-compatible expression vectors for functional analysis and subcellular localization of recombinant proteins. We expressed Arabidopsis ABCB1 and ABCB19 in mam1pdr1 host lines under the inducible nmt41 promoter. ABCB19 showed a higher (3)H-IAA export activity than ABCB1. Arabidopsis PIN proteins were expressed in a mutant lacking the auxin effluxer like 1 (AEL1) gene. PIN1 showed higher activity than PIN2 with similar protein expression levels. Expression of AUX1 in a permease-deficient vat3 mutant resulted in increased net auxin uptake activity. Finally, ABCB4 expressed in mam1pdr1 displayed a concentration-dependent reversal of (3)H-IAA transport that is consistent with its observed activity in planta. Structural modelling suggests that ABCB4 has three substrate interaction sites rather than the two found in ABCB19, thus providing a rationale for the observed substrate activation. Taken together, these results suggest that the S. pombe system described here can be employed for comparative analyses and subsequent structural characterizations of plant transport proteins.
基于烟草BY - 2细胞、拟南芥细胞培养物、非洲爪蟾卵母细胞、酿酒酵母和人HeLa细胞的异源表达系统已被用于表达和鉴定拟南芥中的PIN、ABCB（PGP）和AUX/LAX生长素转运蛋白。然而，尚未找到可用于对这些蛋白质进行有效比较分析的单一系统。我们开发了一种易于使用的粟酒裂殖酵母系统，用于植物转运蛋白的比较研究。该系统包括所有ABC和假定的生长素转运基因的敲除突变体，以及用于重组蛋白功能分析和亚细胞定位的Gateway（R）兼容表达载体。我们在可诱导的nmt41启动子下，在mam1pdr1宿主系中表达了拟南芥ABCB1和ABCB19。ABCB19显示出比ABCB1更高的（3）H - IAA输出活性。拟南芥PIN蛋白在缺乏生长素输出类似物1（AEL1）基因的突变体中表达。在相似的蛋白表达水平下，PIN1的活性高于PIN2。在缺乏通透酶的vat3突变体中表达AUX1导致净生长素摄取活性增加。最后，在mam1pdr1中表达的ABCB4表现出（3）H - IAA转运的浓度依赖性逆转，这与其在植物中观察到的活性一致。结构建模表明，ABCB4有三个底物相互作用位点，而不是ABCB19中的两个，从而为观察到的底物激活提供了理论依据。综上所述，这些结果表明，本文所述的粟酒裂殖酵母系统可用于植物转运蛋白的比较分析和后续的结构表征。
