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

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.

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