A plant Bro1 domain protein BRAF regulates multivesicular body biogenesis and membrane protein homeostasis

Jinbo Shen,Xiangfeng Wang,Yonglun Zeng,Qiong Zhao,Ying Zhu,Caiji Gao,Liwen Jiang

doi:10.1038/s41467-018-05913-y

Abstract

Plant development, defense, and many physiological processes rely on the endosomal sorting complex required for transport (ESCRT) machinery to control the homeostasis of membrane proteins by selective vacuolar degradation. Although ESCRT core components are conserved among higher eukaryotes, the regulators that control the function of the ESCRT machinery remain elusive. We recently identified a plant-specific ESCRT component, FREE1, that is essential for multivesicular body/prevacuolar compartment (MVB/PVC) biogenesis and vacuolar sorting of membrane proteins. Here we identify a plant-specific Bro1-domain protein BRAF, which regulates FREE1 recruitment to the MVB/PVC membrane by competitively binding to the ESCRT-I component Vps23. Altogether, we have successfully identified a role for BRAF, whose function as a unique evolutionary ESCRT regulator in orchestrating intraluminal vesicle formation in MVB/PVCs and the sorting of membrane proteins for degradation in plants makes it an important regulatory mechanism underlying the ESCRT machinery in higher eukaryotes.

Highlights

Plant development, defense, and many physiological processes rely on the endosomal sorting complex required for transport (ESCRT) machinery to control the homeostasis of membrane proteins by selective vacuolar degradation
Through a combination of cellular, biochemical, and genetic approaches, we further demonstrate that BRo1-domain protein As FREE1 suppressor (BRAF) and FREE1 compete for binding to ESCRT-I component Vps[23] on multivesicular body/prevacuolar compartment (MVB/PVC) and function as an important regulator for FREE1 function in intraluminal vesicles (ILVs) formation of MVB/PVC and membrane protein vacuolar sorting
We first identified a plant Bro1-domain protein that regulates the ILVs of MVB formation by demonstrating (1) through both in vitro and in vivo biochemical analysis that BRAF is incorporated into the ESCRT-I complex and competes with FREE1 for binding to Vps[23]; (2) with genetic data, which show that depletion of BRAF increases FREE1 recruitment to MVB/PVCs, providing a rationale for the reversion of FREE1-RNAi phenotypes; and (3) that overexpression of endosome-localized BRAF gives rise to a seedling lethality phenotype as do FREE1 loss-offunction mutants with defects in the formation of ILVs in MVBs leading to the accumulation of ubiquitinated membrane proteins

Summary

Cerulean -Vps28A

Both the in vitro pull-down assay and the in vivo IP assay showed that BRAF(A330V), compared to the WT version, displayed reduced interaction with Vps23A (Fig. 4e–g) Taken together, these results indicate that BRAF competes with FREE1 for binding to Vps[23] and that the alanine-to-valine mutation in sof[524] decreased the competition binding. Further quantification analysis of the three-dimensional (3D) confocal images showed that either YFP-Rha[1] or VHA-a1-GFP exhibited identical patterns with similar numbers of punctae in WT and braf-2 mutant (Supplementary Fig. 8C–E) These results indicate that the increased recruitment of GFP-FREE1 to MVB/ PVCs is caused by depletion of BRAF. These results demonstrated that BRAF is S-acylated for its PM localization, and that the de-acylated BRAF(C3A) mutant is mainly recruited to the MVB/PVCs and interacts with Vps[23]

DEX::FREE1-RNAi

Discussion

Findings

Methods