RST1 is a FREE1 suppressor that negatively regulates vacuolar trafficking in Arabidopsis

FYVE domain protein required for endosomal sorting 1 (FREE1), a plant specific endosomal sorting complex required for transport I (ESCRT) component, is essential for biogenesis of multivesicular bodies (MVBs), vacuolar degradation of membrane protein, cargo vacuolar sorting, autophagic degradation and vacuole biogenesis in Arabidopsis. Here we report the characterization of RST1 as suppressor of free1, when mutated as null mutant, allows the survival of FREE1-RNAi knockdown line, restores the normal MVB and vacuole formation. The RST1 gene encodes an evolutionary conserved multicellular organism specific protein, containing two DUF3730 (Domain of Unknown Function 3730) domains, showing no similarity to known proteins, and predominantly localizes in cytosol. The depletion of FREE1 caused significant accumulation of RST1, and transgenic Arabidopsis plants over-expressing RST1 showed retarded seedling growth with dilated MVB, and inhibition of endocyted FM4-64 dye to vacuole membrane, suggesting a negative role of RST1 in vacuolar transport. Consistently, enhanced endocytic degradation of membrane vacuolar cargos occurred in rst1 mutant. Further transcriptomic comparison of rst1 with free1 revealed a negative correlation of gene expression profiles, demonstrating an antagonistic function of FREE1 and RST1. Thus, RST1 is a negative regulator controlling membrane protein homeostasis and FREE1-mediated functions in plants.