Kifunensine-sensitive ADP-ribosylation factor A1EG69R mutant reveals coordination of protein glycosylation and vesicle transport pathways.

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.