The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis
The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis
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DOI：
10.1073/pnas.1406073111
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发表时间：
2014-08-05
影响因子：
11.1
通讯作者：
Orellana, Ariel
中科院分区：
文献类型：
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作者：
Rautengarten, Carsten;Ebert, Berit;Orellana, Ariel
植物细胞被细胞壁包围，细胞壁在植物生长、结构完整性和防御中起着关键作用。细胞壁是一种复杂多样的结构，主要由多糖组成。大多数非纤维素细胞壁多糖在高尔基体中由主要在细胞质中合成的核苷酸糖产生。这些核苷酸糖从胞质溶胶运输到高尔基体腔是细胞壁生物合成的关键过程，并且由核苷酸糖转运蛋白（NST）家族介导。许多研究试图通过NST表征底物特异性转运;然而，某些底物的可用性和缺乏稳健的方法已被证明是有问题的。因此，我们开发了一种新的方法，将NST重组到脂质体中，并随后通过质谱法评估核苷酸糖的摄取。为了解决底物可用性的限制，我们还开发了一种两步反应，通过表达拟南芥UDP-L-Rha合酶的两个活性结构域来酶促合成UDP-L-鼠李糖（Rha）。采用脂质体方法和新合成的底物对拟南芥NSTs的一个进化枝进行分析，鉴定并鉴定了6个双功能UDP-LRha/UDP-D-半乳糖（Gal）转运蛋白（URGT）。对这些URGT中的两种的功能丧失和过表达植物的进一步分析支持它们在转运UDP-L-Rha和UDP-D-Gal用于基质多糖生物合成中的作用。
Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP-L-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP-L-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP-LRha/UDP-D-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP-L-Rha and UDP-D-Gal for matrix polysaccharide biosynthesis.