VAN3, an Arabidopsis novel type ArfGAP protein, functions in a vesicle transport governing vascular continuity

Abstract

We have cloned the VAN3 gene as a causal gene of van3 mutant, defective in leaf vascular continuity. VAN3 encodes a putative novel type ArfGAP protein in Arabidopsis thaliana. Here we report ArfGAP activity of VAN3, and its subcellular localization.
First, we determined ArfGAP activity of VAN3 by an in vitro GTP/GDP exchange assay. VAN3 protein, which was prepared from E.coli havoring VAN3, stimulated hydrolysis of GTP to GDP binding to yeast Arf1p. Next, we determined the subcellular localization of VAN3 using a fusion protein of VAN3 and Venus. VAN3-Venus expressed under the control of 35S promoter showed fluorescent dots moving around in the cytoplasm. Double labeling experiments using VAN3-Venus and FM4-64, revealed high colocalization of FM4-64 staining vesicles and VAN3-Venus staining dots, suggesting VAN3 locates in the endosomal compartment. Efforts are underway to reveal colocalization of GNOM ArfGEF and VAN3 ArfGAP, and cell specificity of VAN3 gene expression.