Interaction between VPS35 and RABG3f is necessary as a checkpoint to control fusion of late compartments with the vacuole Cecilia Rodriguez-Furlana,b, David Domozychc, Weixing Qiand,e, Per-Anders Enquistd,e, Xiaohui Lif,g, Chunhua Zhangf,g, Rolf Schenka,b, Holly Saulsbery Winbiglerh, William Jacksonh, Natasha V. Raikhela,b, and Glenn R. Hicksa,b,i,1 aDepartment of Botany and Plant Sciences, University of California, Riverside, CA 92506; bInstitute of Integrative Genome Biology, University of California, Riverside, CA 92506; cBiology Department, Skidmore College, Saratoga Springs, NY 12866; dDepartment of Chemistry, Umea University, SE-901 87 Umea, Sweden; eDepartment of Chemistry, Chemical Biology Consortium Sweden, SE-901 87 Umea, Sweden; fDepartment of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47906; gCenter for Plant Biology, Purdue University, West Lafayette, IN 47906; hDepartment of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 20201; and iUppsala BioCenter, Swedish University of Agricultural Sciences, SE-75007 Uppsala, Sweden Edited by Niko Geldner, University of Lausanne, Lausanne, Switzerland, and accepted by Editorial Board Member Joseph R. Ecker September 5, 2019 (received for review March 28, 2019) Vacuoles are essential organelles in plants, playing crucial roles, vesicular bodies (MVBs) that ultimately fuse with the vacuole such as cellular material degradation, ion and metabolite storage, membrane (tonoplast) (6). The specificity of vesicular transport and turgor maintenance. Vacuoles receive material via the endo- relies mainly on RAB-GTPases that coordinate the machineries cytic, secretory, and autophagic pathways. Membrane fusion is the for endosomal maturation, trafficking, and membrane fusion (7). last step during which prevacuolar compartments (PVCs) and RABs function as molecular switches that exist in an inactive autophagosomes fuse with the vacuole membrane (tonoplast) to GDP-bound form and an active GTP-bound form. Upon GTP deliver cargoes. Protein components of the canonical intracellular loading via a membrane-localized guanine nucleotide exchange Ara- fusion machinery that are conserved across organisms, including factor (GEF), RABs interact with effectors, such as specific sub- bidopsis thaliana, include complexes, such as soluble N-ethylmaleimide– units of vesicle tethering complexes. These complexes function as PLANT BIOLOGY sensitive factor attachment protein receptors (SNAREs), that cata- adaptors between vesicles and target membranes, which assists in lyze membrane fusion, and homotypic fusion and vacuole protein the assembly of membrane-bound soluble N-ethylmaleimide– sorting (HOPS), that serve as adaptors which tether cargo vesicles sensitive factor attachment protein receptors (SNAREs) into a to target membranes for fusion under the regulation of RAB- GTPases. The mechanisms regulating the recruitment and assem- stable 4-helix bundle that catalyzes fusion of the 2 lipid bilayers (8). The mechanisms of endosomal maturation to form PVCs/ bly of tethering complexes are not well-understood, especially MVBs and fusion with the vacuole are well-studied in yeast but the role of RABs in this dynamic regulation. Here, we report the have not been fully elucidated in plants where there is greater identification of the small synthetic molecule Endosidin17 (ES17), redundancy of genes encoding many trafficking components, which interferes with synthetic, endocytic, and autophagic traffic including RABs (9, 10). In Arabidopsis during late trafficking to 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 teth- Significance ering complex, preventing its normal interaction with the Arabidopsis RAB7 homolog RABG3f. ES17 interference with VPS35–RABG3f in- In plants, the retromer complex (VPS35, VPS29, VPS26) has been teraction prevents the retromer complex to endosome anchoring, implicated in retrograde traffic from the endosomes back to the resulting in retention of RABG3f. Using multiple approaches, we trans-Golgi network. Nevertheless, vps35 mutants exhibit im- show that VPS35–RABG3f–GTP interaction is necessary to trigger paired anterograde traffic toward the vacuole (synthetic, endo- downstream events like HOPS complex assembly and fusion of late cytic, and autophagic). This paradox remains an open question. compartments with the tonoplast. Overall, our results support a role We show that the small molecule endosidin17 (ES17) targets for the interaction of RABG3f–VPS35 as a checkpoint in the con- VPS35, which disrupts its association with RABG3f. This, in turn, trol of traffic toward the vacuole. impairs retromer complex membrane anchoring, producing vps35 mutant-like phenotypes. Using ES17, we found that retromer | small molecule | RAB7 | RABG3f | VPS35 VPS35–RABG3f binding precedes the release of RABG3f–GTP to interact with the vacuolar homotypic fusion and vacuole protein n plant cells, vacuoles are essential organelles that maintain cellular sorting (HOPS) complex, which is required for fusion of late – Ihomeostasis, serving as reservoirs for multiple molecules and compartments with the tonoplast. Thus, VPS35 RABG3f inter- acting as lytic organelles for the breakdown and recycling of action constitutes a checkpoint before proceeding with cellular components, among others. Therefore, vacuoles are membrane fusion. central for multiple processes, like cell elongation, programmed Author contributions: C.R.-F., P.-A.E., W.J., N.V.R., and G.R.H. designed research; C.R.-F., cell death, and sequestration of toxic compounds, among others. D.D., W.Q., P.-A.E., X.L., C.Z., R.S., H.S.W., and W.J. performed research; C.R.-F., W.Q., These diverse functions are fulfilled through tight regulation of P.-A.E., C.Z., N.V.R., and G.R.H. contributed new reagents/analytic tools; C.R.-F., X.L., endomembrane trafficking to the vacuoles. Three endomem- R.S., and W.J. analyzed data; and C.R.-F. wrote the paper. brane pathways are known to direct proteins to the vacuole: the The authors declare no competing interest. endocytic, synthetic, and macroautophagic (hereafter auto- This article is a PNAS Direct Submission. N.G. is a guest editor invited by the phagic) routes. Proteins synthesized in the endoplasmic reticulum Editorial Board. (ER) can be transported to the vacuole using a Golgi-dependent Published under the PNAS license. or a Golgi-independent synthetic pathway (1–4). Post-Golgi 1To whom correspondence may be addressed. Email: [email protected]. synthetic traffic and endocytic traffic merge at the trans-Golgi This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. network (TGN)/early endosomes (EEs) (5). TGN/EE compart- 1073/pnas.1905321116/-/DCSupplemental. ments mature into prevacuolar compartments (PVCs) or multi- www.pnas.org/cgi/doi/10.1073/pnas.1905321116 PNAS Latest Articles | 1of11 Downloaded by guest on September 29, 2021 the vacuole, PVCs initially interact with RAB5 GTPases that Our results reveal a previously uncharacterized function for become activated by the GEF protein VPS9; RAB5s then inter- the interaction of VPS35–RABG3f in plants in which the com- act with the class C core vacuole/endosome tethering (CORVET) plex acts as a checkpoint preceding tonoplast fusion. complex composed of the subunits VPS8, VPS3, VPS11, VPS16, VPS18, and VPS33 (11). CORVET permits PVC in- Results teraction with the SNAREs VAMP727 and SYP22. This ulti- Perturbing Vacuole Traffic Pharmacologically to Understand Regulation. mately results in the fusion of RAB5-positive PVCs with the To gain insight about the mechanisms regulating vacuolar traffic, we tonoplast. Additionally, the recruitment of the Mon1–Ccz1 GEF reasoned that drug-like molecules that interfere with late trafficking complex triggers the exchange of RAB5 for RAB7 (RABG3 to the vacuole, where the synthetic and endocytic autophagic family members in Arabidopsis) and catalyzes the activation of pathways merge, would include protein targets shared among the RAB7 (12–15). This exchange in RAB appears to determine the pathways that are involved in their coordinate regulation. specificity of recruitment of sorting machineries via tethering For a small molecule screen, we took advantage of the be- complexes and fusion. The retromer is one such tethering com- havior of the membrane marker protein PIN2-GFP at the epi- plex formed by the VPS35, VPS26, and VPS29 subunits which dermal cells of the root, which is endocytosed and trafficked to mediates recycling of transmembrane cargo receptors from the lytic vacuole in response to dark treatment. Under dark PVCs and tonoplast back to the TGN (14). The RAB7 family conditions, GFP is more stable in lytic vacuoles due to confor- member RABG3f is required for endosomal recycling and binds mational changes allowing its visualization (24, 25). Using the directly to VPS35 (16). RABG3f also interacts with the vacuolar Endosidin chemical collection (26), we selected Endosidin17 homotypic fusion and vacuole protein sorting (HOPS) tethering (ES17), which inhibited the vacuole accumulation of PIN2-GFP in the dark (Fig. 1A). After ES17 treatment, PIN2-GFP accu- complex, which is required for fusion of PVCs with the tonoplast mulated in cytoplasmic structures that colocalized with the (11). HOPS is composed of the subunits VPS41, VPS39, VPS33, endocytic marker FM4-64 and the TGN/PVC marker ARA7- VPS11, VPS18,