Interaction between VPS35 and RABG3f is necessary as a checkpoint to control fusion of late compartments with the vacuole

Abstract

Vacuoles are essential organelles in plants, playing crucial roles, such as cellular material degradation, ion and metabolite storage, and turgor maintenance. Vacuoles receive material via the endocytic, secretory, and autophagic pathways. Membrane fusion is the last step during which prevacuolar compartments (PVCs) and autophagosomes fuse with the vacuole membrane (tonoplast) to deliver cargoes. Protein components of the canonical intracellular fusion machinery that are conserved across organisms, including Arabidopsis thaliana, include complexes, such as soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), that catalyze membrane fusion, and homotypic fusion and vacuole protein sorting (HOPS), that serve as adaptors which tether cargo vesicles to target membranes for fusion under the regulation of RAB-GTPases. The mechanisms regulating the recruitment and assembly of tethering complexes are not well-understood, especially the role of RABs in this dynamic regulation. Here, we report the identification of the small synthetic molecule Endosidin17 (ES17), which interferes with synthetic, endocytic, and autophagic traffic by impairing the fusion of late endosome compartments with the tonoplast. Multiple independent target identification techniques revealed that ES17 targets the VPS35 subunit of the retromer tethering complex, preventing its normal interaction with the Arabidopsis RAB7 homolog RABG3f. ES17 interference with VPS35–RABG3f interaction prevents the retromer complex to endosome anchoring, resulting in retention of RABG3f. Using multiple approaches, we show that VPS35–RABG3f–GTP interaction is necessary to trigger downstream events like HOPS complex assembly and fusion of late compartments with the tonoplast. Overall, our results support a role for the interaction of RABG3f–VPS35 as a checkpoint in the control of traffic toward the vacuole.

Rab22a is a novel prognostic marker for cell progression in breast cancer

“…A set of Rab proteins have been reported to promote tumor aggression by regulating intracellular signaling. The signaling pathway identified by GSEA is consistent with that of other published studies (35)(36)(37). Overexpression of Rab1A promotes colorectal cancer progression and invasion by activating mTorc1 signaling.…”

The regulatory role of the Aspergillus flavus core retromer complex in aflatoxin metabolism

“…7 E ). In previous studies, a small molecule compound (ES17) effectively blocked the interaction between Vps35 and Rab7 and prevented the late fusion of vacuoles in Arabidopsis ( 40 ). In this study, ES17 at a high concentration (above 250 μM) promoted the level of aflatoxin and inhibited the growth of fungal colonies ( Figs.…”

“…In previous reports, a Vps35 partner Ypt7 was necessary for vesicle-vacuole fusion and aflatoxin increase in Aspergillus nidulans ( 20 ). In Arabidopsis , the interaction of Vps35 homolog of Rab7 was necessary for triggering the fusion of vacuoles ( 40 ). We found that knocking-out the Aflvps35 gene in A. flavus also led to the fragmentation of the vacuoles ( Fig.…”

“…This effect of ES17 in A. flavus was similar to the deletion of AflVps35 or AflRab7. In addition, ubiquitinated proteins in autophagosomes of Arabidopsis accumulated under ES17 treatment ( 40 ). Therefore, we examined the ubiquitination levels in WT and Aflvps35 deletion mutants and found that the level of total ubiquitination in the Δ Aflvps35 mutant was much higher than that in the WT strain ( Fig.…”

“…In Arabidopsis , Rabg3f (the Rab7 homologous family) is necessary for endosomal circulation and directly binds to Vps35 ( 34 ). First, Vps35–Rabg3f interaction is necessary for the complex assembly of HOPS and the fusion of late vacuoles as a checkpoint in the control of traffic toward the vacuole ( 40 ). Vps41 (HOPS complex subunit)-Rab7 then mediates the fusion of late endosomes/vacuoles ( 41 ).…”