Brassinosteroid (BR) hormone signaling controls multiple processes during plant growth and development and is initiated at the plasma membrane through the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) together with co-receptors such as BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1).BRI1 abundance is regulated by endosomal recycling and vacuolar targeting,but the role of vacuole-related proteins in BR receptor dynamics and BR responses remains elusive.Here,we show that the absence of two DUF300 domain-containing tonoplast proteins,LAZARUS1 (LAZ1) and LAZ1 HOMOLOG1 (LAZ1H1),causes vacuole morphology defects,growth inhibition,and constitutive activation of BR signaling.Intriguingly,tonoplast accumulation of BAK1 was substantially increased and appeared causally linked to enhanced BRI1 trafficking and degradation in laz1 laz1h1 plants.Since unrelated vacuole mutants exhibited normal BR responses,our findings indicate that DUF300 proteins play distinct roles in the regulation of BR signaling by maintaining vacuole integrity required to balance subcellular BAK1 pools and BR receptor distribution.