Storage protein is the most abundant nutritional component in soybean seed. Morphology-based evidence has verified that storage proteins are initially synthesized on the endoplasmic reticulum, and then follow the Golgi-mediated pathway to the protein storage vacuole. However, the molecular mechanisms of storage protein trafficking in soybean remain unknown. Here, we clone the soybean homologs of Rab5 and its guanine nucleotide exchange factor (GEF) VPS9. GEF activity combined with yeast two-hybrid assays demonstrated that GmVPS9a2 might specifically act as the GEF of the canonical Rab5, while GmVPS9b functions as a common activator for all Rab5s. Subcellular localization experiments showed that GmRab5a was dually localized to the trans-Golgi network and pre-vacuolar compartments in developing soybean cotyledon cells. Expression of a dominant negative variant of Rab5a, or RNAi of either Rab5a or GmVPS9s, significantly disrupted trafficking of mRFP-CT10, a cargo marker for storage protein sorting, to protein storage vacuoles in maturing soybean cotyledons. Together, our results systematically revealed the important role of GmRab5a and its GEFs in storage protein trafficking, and verified the transient expression system as an efficient approach for elucidating storage protein trafficking mechanisms in seed.

中文翻译：

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小GTPase Rab5a及其鸟嘌呤核苷酸交换因子参与高尔基后发育中的大豆子叶贮藏蛋白的运输。

贮藏蛋白是大豆种子中最丰富的营养成分。基于形态学的证据已证明，存储蛋白最初是在内质网上合成的，然后遵循高尔基体介导的途径到达蛋白存储液泡。然而，大豆中贮藏蛋白运输的分子机制仍是未知的。在这里，我们克隆了Rab5的大豆同源物及其鸟嘌呤核苷酸交换因子（GEF）VPS9。GEF活性与酵母双杂交检测相结合表明，GmVPS9a2可能专门充当经典Rab5的GEF，而GmVPS9b充当所有Rab5的共同激活剂。亚细胞定位实验表明，GmRab5a双重定位于发育中的大豆子叶细胞中的反式高尔基网络和前真空区室。Rab5a或Rab5a或GmVPS9s的RNAi的显性阴性变异体的表达显着破坏了mRFP-CT10的运输，这是一种用于存储蛋白质分拣的货物标记，向成熟的大豆子叶中的蛋白质存储空泡转移。在一起，我们的结果系统地揭示了GmRab5a及其GEF在存储蛋白运输中的重要作用，并验证了瞬时表达系统是阐明种子中存储蛋白运输机制的有效方法。