Identification of a vacuolar sucrose transporter in barley and arabidopsis mesophyll cells by a tonoplast proteomic approach
Identification of a vacuolar sucrose transporter in barley and arabidopsis mesophyll cells by a tonoplast proteomic approach
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DOI：
10.1104/pp.106.079533
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发表时间：
2006-05-01
期刊：
影响因子：
7.4
通讯作者：
Schmidt, UG
中科院分区：
文献类型：
--
作者：
Endler, A;Meyer, S;Schmidt, UG
液泡是主要的细胞储存池，蔗糖在液泡中积累到高浓度。虽然有限数量的液泡膜蛋白，如v型H+- atp酶和H+-焦磷酸酶，已经被很好地表征，但大多数液泡转运蛋白仍然是未知的，其中包括负责液泡吸收和释放的转运蛋白。为了寻找新的媒质转运体，我们采用蛋白质组学方法，分析了大麦（Hordeum vulgare）高纯度叶肉液泡的媒质部分。我们鉴定了101种蛋白质，包括88种空泡蛋白和假定的空泡蛋白。SUT转运蛋白HvSUT2是在40个空泡蛋白中发现的，此前在拟南芥（Arabidopsis thaliana）空泡蛋白组学研究中未报道。为了确认这种转运体在细胞质中的定位，我们构建并表达了绿色荧光蛋白（GFP）与HvSUT2及其最接近的拟南芥同源物AtSUT4的融合蛋白。在拟南芥叶片和洋葱表皮细胞中瞬时表达HvSUT2-GFP和AtSUT4-GFP，可在叶绿体上产生绿色荧光，表明这些转运蛋白确实位于液泡膜上。利用微毛细管，我们从叶片原生质体制备中选择了叶肉原生质体，并明确证明，与伴生细胞特异性的atsu2不同，HvSUT2和atsu4在叶肉原生质体中表达，这表明HvSUT2和atsu4参与了光合作用衍生的硫的运输和液泡储存。
The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H+-ATPases and H+-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.