Nagashima Yukihiro, Sharma Vinita, Reekers Lea-Franziska, von Schaewen Antje, Koiwa Hisashi
Vegetable and Fruit Improvement Center and Department of Horticultural Sciences Texas A&M University, College Station, TX 77843, USA.
Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, D-48149 Münster, Germany.
J Exp Bot. 2025 May 27;76(8):2112-2128. doi: 10.1093/jxb/eraf017.
Complex N-glycans are asparagine (N)-linked branched sugar chains attached to secretory proteins in eukaryotes. They are produced by modification of N-linked oligosaccharide structures in the endoplasmic reticulum and Golgi apparatus. Complex N-glycans formed in the Golgi apparatus are often assigned specific roles unique to the host organism, with their roles in plants remaining largely unknown. Using inhibitor (kifunensine, KIF) hypersensitivity as read out, we identified Arabidopsis mutants that require complex N-glycan modification. Among >100 KIF-sensitive mutants, one showing abnormal secretory organelles and a salt-sensitive phenotype contained a point mutation leading to amino acid replacement (G69R) in ARFA1E, a small Arf1-GTPase family protein presumably involved in vesicular transport. In vitro assays showed that the G69R exchange interferes with protein activation. In vivo, ARFA1EG69R caused dominant-negative effects, altering the morphology of the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network (TGN). Post-Golgi transport (endocytosis/endocytic recycling) of the essential glycoprotein KORRIGAN1, one of the KIF sensitivity targets, is slowed down constitutively as well as under salt stress in the ARFA1EG69R mutant. Because regulated cycling of plasma membrane proteins is required for stress tolerance of the host plants, the ARFA1EG69R mutant established a link between KIF-targeted luminal glycoprotein functions/dynamics and cytosolic regulators of vesicle transport in endosome-/cell wall-associated tolerance mechanisms.
复杂N-聚糖是真核生物中与分泌蛋白相连的天冬酰胺（N）连接的分支糖链。它们是通过在内质网和高尔基体中对N-连接寡糖结构进行修饰而产生的。在高尔基体中形成的复杂N-聚糖通常被赋予宿主生物体特有的特定功能，而它们在植物中的作用在很大程度上仍不清楚。我们以抑制剂（ kifunensine，KIF）超敏反应为指标，鉴定了需要复杂N-聚糖修饰的拟南芥突变体。在100多个对KIF敏感的突变体中，有一个显示出分泌细胞器异常和盐敏感表型，其在ARFA1E中含有一个导致氨基酸替换（G69R）的点突变，ARFA1E是一种可能参与囊泡运输的小Arf1-GTPase家族蛋白。体外实验表明，G69R替换会干扰蛋白质激活。在体内，ARFA1EG69R会产生显性负效应，改变内质网、高尔基体和反式高尔基体网络（TGN）的形态。作为KIF敏感性靶点之一的必需糖蛋白KORRIGAN1在高尔基体后运输（内吞作用/内吞循环）在ARFA1EG69R突变体中，无论是在正常情况下还是在盐胁迫下都会持续减慢。由于宿主植物的胁迫耐受性需要质膜蛋白的调节循环，ARFA1EG69R突变体在内体/细胞壁相关耐受机制中建立了KIF靶向的腔内糖蛋白功能/动力学与囊泡运输的胞质调节因子之间的联系。
