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

Abstract

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 (ER) 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 over 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 ER, Golgi apparatus, and trans-Golgi network (TGN). Post-Golgi transports (endocytosis/endocytic recycling) of essential glycoprotein KORRIGAN1, one of KIF-sensitivity targets, is slowed down constitutively as well as under salt stress in ARFA1EG69R mutant. Because regulated cycling of plasma membrane proteins is required for stress tolerance of the host plants, 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.