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Phosphoinositides Control the Localization of HOPS Subunit VPS41, Which Together with VPS33 Mediates Vacuole Fusion in Plants

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Phosphoinositides Control the Localization of HOPS Subunit VPS41, Which Together with VPS33 Mediates Vacuole Fusion in Plants Phosphoinositides control the localization of HOPS subunit VPS41, which together with VPS33 mediates vacuole fusion in plants Carla Brilladaa,1, Jiameng Zhenga,1,2, Falco Krügerb, Eliezer Rovira-Diaza,3, Jana Christin Askanib, Karin Schumacherb, and Marcela Rojas-Piercea,4 aDepartment of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695; and bCentre for Organismal Studies, Plant Developmental Biology, Heidelberg University, 69120 Heidelberg, Germany Edited by Natasha V. Raikhel, Center for Plant Cell Biology, Riverside, CA, and approved July 19, 2018 (received for review May 16, 2018) The vacuole is an essential organelle in plant cells, and its dynamic vacuolar soluble NSF attachment protein receptor (SNARE) nature is important for plant growth and development. Homotypic complex containing VTI11 (2). membrane fusion is required for vacuole biogenesis, pollen germi- Vacuole fusion is not well characterized in plants, but in nation, stomata opening, and gravity perception. Known compo- eukaryotic cells it requires SNAREs, RAB GTPases, membrane nents of the vacuole fusion machinery in eukaryotes include SNARE lipids, and a tethering complex. SNARE proteins mediate proteins, Rab GTPases, phosphoinositides, and the homotypic fusion membrane fusion by assembly of a four-helix bundle, or trans- and vacuolar protein sorting (HOPS) tethering complex. HOPS SNARE complex, from one SNARE located in the incoming function is not well characterized in plants, but roles in embryo- vesicle and three SNAREs located in the target membrane. The genesis and pollen tube elongation have been reported. Here, we SNARE complex brings the two membranes together via a zip- show that Arabidopsis HOPS subunits VPS33 and VPS41 accumulate pering mechanism that is thought to result in membrane fusion in late endosomes and that VPS41, but not VPS33, accumulates (16). A SNARE complex for plant prevacuolar compartments (PVCs) and the vacuole is composed of SYP22/VAM3 (Qa- in the tonoplast via a wortmannin-sensitive process. VPS41 and SNARE), VTI11 (Qb-SNARE, called Vti1p in yeast), SYP51 VPS33 proteins bind to liposomes, but this binding is inhibited by (Qc-SNARE), and VAMP727 (R-SNARE) (17, 18). The Qabc PLANT BIOLOGY phosphatidylinosiltol-3-phosphate [PtdIns(3)P] and PtdIns(3,5)P2, complex can also associate with the R-SNAREs VAMP711 or which implicates a nonconserved mechanism for HOPS recruitment VAMP713 (19, 20). A role of these SNAREs in vacuole fusion is in plants. Inducible knockdown of VPS41 resulted in dramatic vacu- evidenced by altered vacuole phenotypes of vti11,syp22, and ole fragmentation phenotypes and demonstrated a critical role for syp22 vamp727 double mutants (2, 18, 21). HOPS in vacuole fusion. Furthermore, we provide evidence for ge- The homotypic fusion and vacuole protein sorting (HOPS) netic interactions between VPS41 and VTI11 SNARE that regulate tethering complex was recently shown to mediate homotypic vac- vacuole fusion, and the requirement of a functional SNARE complex uole fusion in plants (3). HOPS is important for the fusion of for normal VPS41 and VPS33 localization. Finally, we provide evi- vacuoles in yeast (16, 22), and fusion of lysosomes in Caenorhabditis dence to support VPS33 and SYP22 at the initial stage for HOPS– SNARE interactions, which is similar to other eukaryotes. These Significance results highlight both conserved and specific mechanisms for HOPS recruitment and function during vacuole fusion in plants. Plant vacuoles are essential organelles and occupy up to 90% of the cell volume. Their roles include regulation of stomata move- Arabidopsis | vacuole fusion | HOPS | SNARE | phosphoinositides ments, protein storage in seeds, gravity sensing, and ion ho- meostasis. Vacuole or lysosome fusion in eukaryotes is mediated he plant vacuole is an essential organelle with multiple roles by two multisubunit complexes, SNARE and homotypic fusion and Tincluding storage, pH homeostasis, and recycling (1). Vacu- vacuolar protein sorting (HOPS), but only the SNARE complex is ole fusion is required for stomata opening, pollen fertility, and well characterized in plants. Here, we show that, similar to other embryo viability (2–4). Two types of vacuoles exist in plant cells. eukaryotes, HOPS mediates vacuole fusion in plants by interaction Embryos in developing seeds contain multiple protein storage with SNAREs and that the HOPS subunit VPS33 and the SNARE vacuoles (PSVs), which have a neutral pH and accumulate protein SYP22 display the sites for interaction between these storage proteins such as the 7S and 11S globulins. Cells in most complexes. In contrast to other eukaryotes, however, plant HOPS mature tissues, instead, contain one large lytic vacuole, which is recruitment to liposomes is inhibited by phosphoinositides, which acidic, abundant in lytic proteases, and critical for ion homeo- appear to define strict rules for regulating fusion and fragmen- stasis and cell turgor (5). Transition from PSVs to lytic vacuoles tation of dynamic vacuoles. occurs during germination (6) and involves PSV fusion and Author contributions: C.B., J.Z., F.K., J.C.A., K.S., and M.R.-P. designed research; C.B., J.Z., maturation (7). Vacuoles in root meristematic cells are formed F.K., E.R.-D., J.C.A., and M.R.-P. performed research; C.B., J.Z., F.K., K.S., and M.R.-P. ana- by interconnected tubular structures (8), and membrane fusion is lyzed data; and C.B. and M.R.-P. wrote the paper. necessary for the establishment of the central lytic vacuole (2). The authors declare no conflict of interest. Transient and mobile structures such as transvacuolar strands, This article is a PNAS Direct Submission. sheets, and bulbs contribute to the dynamic nature of plant vacuoles Published under the PNAS license. – (9 12). In vegetative cells, however, very little vacuole fragmenta- 1C.B. and J.Z. contributed equally to this work. tion occurs and most cells contain a single large vacuole. Two ex- 2Present address: Product Department, Abmart, Shanghai 200233, China. ceptions are the changes in guard cell vacuoles during stomata 3Present address: Department of Microbiology, Biochemistry and Molecular Genetics, movements (2, 13, 14) and in the leaf motor cells of the mimosa tree Rutgers, The State University of New Jersey, Newark, NJ 07103. (Albizia julibrissin) (15). In both cases, vacuole remodeling occurs 4To whom correspondence should be addressed. Email: [email protected]. during dramatic changes in cellular turgor that drive organ move- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ments. The changes in guard cell vacuoles that occur during stomata 1073/pnas.1807763115/-/DCSupplemental. opening require homotypic membrane fusion mediated by the www.pnas.org/cgi/doi/10.1073/pnas.1807763115 PNAS Latest Articles | 1of10 Downloaded by guest on September 23, 2021 elegans (23) and humans (24). The yeast HOPS complex is the best Results characterized and is formed by six subunits. Vps39p and Vps41p VPS41 and VPS33 Partially Colocalize with Late Endosomes, but only are HOPS specific, while Vps11p, Vps16p, Vps18p, and Vps33p VPS41 Is Detected at the Tonoplast. VPS41 (AT1G08190) and form the “CORE” and are shared with CORVET, a tethering VPS33 (AT3G54860) share 23.29% and 19.17% sequence identity complex that functions at the early endosome (25, 26). HOPS with yeast Vps41p and Vps33p, respectively. Therefore, VPS33 and recruitment to yeast vacuole membranes requires phosphatidyli- VPS41 are likely homologs of these yeast HOPS proteins (31, 50). nositol 3-phosphate [PtdIns(3)P] and PtdIns(4,5)P2, two important We first investigated whether VPS41 and VPS33 colocalized in regulatory lipids in vacuole fusion (27). Vps33 belongs to the vacuolar or prevacuolar membranes, as expected for HOPS. family of Sec1/Munc18 (SM) proteins, which are implicated in Transient expression of mRFP-VPS41 and VPS33-GFP under assembly and regulation of SNARE complexes. SM proteins have control of UBQ10 promoters in Nicotiana benthamiana leaves a three-domain architecture, in which domains 1 and 3 form a cleft indicated that these proteins accumulate in the cytosol and nu- that accommodates the SNAREs (28). The X-ray structure of a cleus, and associate with membranes in punctate structures (SI fungal Vps33 bound to a single SNARE, together with in vitro fu- Appendix, Fig. S1 A and B). RFP-VPS41 and VPS33-GFP colo- sion assays, suggests that VPS33 catalyzes SNARE complex as- calized in the majority of the punctate structures and the nucleus, sembly through a specific SNARE motif recognition (29). All other and partially at the cell periphery (SI Appendix,Fig.S1C), which HOPS subunits are predicted to form a β-propeller at the N ter- supports their participation in a complex. These experiments, however, do not provide enough resolution to identify the com- minus and an α-solenoid at the C terminus (30). The complete partments where these proteins accumulate and they may be HOPS complex has been detected in Arabidopsis by mass spec- influenced by overexpression. To better characterize protein local- trometry (3), and all subunits are encoded in the Arabidopsis ge- izations, VPS41-GFP (4) and VPS33-GFP were analyzed in stable nomeassinglegenes(31).VACUOLELESS1 (VCL1), the homolog Arabidopsis transgenic plants using full-length genomic fusions to of yeast Vps16, VPS11, VPS39,andVPS41 are required for vacuole GFP under native promoters. Both proteins were again detected in biogenesis and pollen development (3, 4, 32–35). VCL1 interacts the cytosol, but membrane association was more discernible. Similar with VPS33 and VPS11 and localizes to PVCs and the tonoplast to its localization in pollen (4), VPS41-GFP was detected at the (35). VPS18 and VPS39 were also localized to a subdomain of the tonoplast and punctate organelles in roots (Fig. 1A), and this lo- tonoplast and to cytosolic organelles (3). The molecular mecha- calization was not affected by the location of the fluorescence tag as nisms for HOPS function and regulation during vacuole fusion in the localization of YFP-VPS41 was very similar (SI Appendix,Fig.
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