Gahrtz M, Stolz J, Sauer N
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Germany.
Plant J. 1994 Nov;6(5):697-706. doi: 10.1046/j.1365-313x.1994.6050697.x.
In this paper the cloning of a full-length cDNA clone encoding the PmSUC2 sucrose-H+ symporter from Plantago major is described. This plant allows the simple preparation of vascular bundles from the basal regions of fully developed source leaves and thus a separation of vascular and non-vascular tissue. A cDNA library was constructed from poly(A)+ RNA isolated from vascular bundles and used for the subsequent cloning of cDNAs. The respective mRNA is specifically expressed in the vascular bundles as shown on Northern blots of total RNA from vascular and non-vascular tissues. The PmSUC2 protein has 12 putative transmembrane helices and is highly homologous to other plant sucrose transporters. Substrate specificity and energy dependence of the transporter encoded by this cDNA were determined by expression in baker's yeast Saccharomyces cerevisiae. The PmSUC2 protein catalyses the transport of sucrose into transgenic yeast cells. Invertase null mutants of yeast expressing PmSUC2 accumulate sucrose more than 200-fold. This transport was sensitive to uncouplers or SH-group inhibitors. Plasma membranes from yeast cells expressing the PmSUC2 protein were purified and fused to proteoliposomes containing cytochrome-c-oxidase. In this system sucrose is accumulated only when proton motive force is generated, indicating that PmSUC2 is a sucrose-H+ symporter. The apparent molecular weight of the PmSUC2 protein is 35 kDa on 10% SDS-polyacrylamide gels. The presented data strongly support the theory of phloem loading from the apoplastic space by a sucrose-H+ symporter.
本文描述了从大车前草中克隆编码PmSUC2蔗糖-H⁺共转运体的全长cDNA克隆。这种植物能够从完全发育的源叶基部区域简单地制备维管束，从而实现维管组织和非维管组织的分离。从维管束中分离的poly(A)⁺RNA构建了一个cDNA文库，并用于随后的cDNA克隆。如维管组织和非维管组织总RNA的Northern印迹所示，相应的mRNA在维管束中特异性表达。PmSUC2蛋白有12个推定的跨膜螺旋，与其他植物蔗糖转运体高度同源。通过在酿酒酵母中表达来确定该cDNA编码的转运体的底物特异性和能量依赖性。PmSUC2蛋白催化蔗糖转运到转基因酵母细胞中。表达PmSUC2的酵母转化酶缺失突变体积累的蔗糖比正常情况多200倍以上。这种转运对解偶联剂或SH基团抑制剂敏感。纯化表达PmSUC2蛋白的酵母细胞的质膜，并与含有细胞色素c氧化酶的蛋白脂质体融合。在这个系统中，只有当产生质子动力时蔗糖才会积累，这表明PmSUC2是一种蔗糖-H⁺共转运体。在10% SDS-聚丙烯酰胺凝胶上，PmSUC2蛋白的表观分子量为35 kDa。所提供的数据有力地支持了韧皮部通过蔗糖-H⁺共转运体从质外体空间装载物质的理论。
