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SAC Phosphoinositide Phosphatases at the Tonoplast Mediate Vacuolar Function in Arabidopsis

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SAC Phosphoinositide Phosphatases at the Tonoplast Mediate Vacuolar Function in Arabidopsis SAC phosphoinositide phosphatases at the tonoplast mediate vacuolar function in Arabidopsis Petra Novákováa,b,c, Sibylle Hirschb,c,1, Elena Ferarub,c,2, Ricardo Tejosb,c,3, Ringo van Wijkd, Tom Viaeneb,c, Mareike Heilmanne, Jennifer Lerchee, Riet De Ryckeb,c, Mugurel I. Ferarub,c,2, Peter Gronesa,b,c, Marc Van Montagub,c,4, Ingo Heilmanne, Teun Munnikd, and Jirí Frimla,b,c,4 aInstitute of Science and Technology Austria, 3400 Klosterneuburg, Austria; bDepartment of Plant Systems Biology, Flanders Institute for Biotechnology and cDepartment of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; dSection of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, 1090 GE, Amsterdam, The Netherlands; and eDepartment of Cellular Biochemistry, Institute for Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, 06099 Halle, Germany Contributed by Marc Van Montagu, January 2, 2014 (sent for review December 4, 2013) Phosphatidylinositol (PtdIns) is a structural phospholipid that can function of this minor phospholipid. Recent observations also be phosphorylated into various lipid signaling molecules, desig- hint at a role for PPIs in plant vacuoles (18–20), but the data are nated polyphosphoinositides (PPIs). The reversible phosphoryla- scarce and remain inconclusive. tion of PPIs on the 3, 4, or 5 position of inositol is performed by Advances in deciphering various cellular roles of PIs include a set of organelle-specific kinases and phosphatases, and the insights into the phosphatases responsible for hydrolyzing PPIs. characteristic head groups make these molecules ideal for regu- A group of phosphatases, designated suppressor of actin (SAC) lating biological processes in time and space. In yeast and domain phosphatases, has been identified in lower eukaryotes, mammals, PtdIns3P and PtdIns(3,5)P2 play crucial roles in traffick- mammals, and plants (21). Whereas yeast and humans have only ing toward the lytic compartments, whereas the role in plants is five genes harboring the SAC domain, the genome of the model not yet fully understood. Here we identified the role of a land plant Arabidopsis thaliana contains nine genes, of which some plant-specific subgroup of PPI phosphatases, the suppressor of have been functionally characterized and demonstrated to be actin 2 (SAC2) to SAC5, during vacuolar trafficking and morpho- involved in the regulation of stress responses (22–24), polarized Arabidopsis thaliana – genesis in . SAC2 SAC5 localize to the tono- root hair expansion (3), or cell wall formation (25). PLANT BIOLOGY plast along with PtdIns3P, the presumable product of their activ- Here we show that the functionally uncharacterized group of ity. In SAC gain- and loss-of-function mutants, the levels of PtdIns Arabidopsis SAC2–SAC5 proteins that is orthologous to the yeast monophosphates and bisphosphates were changed, with opposite Fig4p is involved in PPI metabolism. SAC2–SAC5 localize along effects on the morphology of storage and lytic vacuoles, and the trafficking toward the vacuoles was defective. Moreover, multiple Significance sac knockout mutants had an increased number of smaller storage and lytic vacuoles, whereas extralarge vacuoles were observed in the overexpression lines, correlating with various growth and de- Polyphosphoinositides (PPIs) are derivatives of the membrane velopmental defects. The fragmented vacuolar phenotype of sac lipid phosphatidylinositol that occur in minor amounts in eu- mutants could be mimicked by treating wild-type seedlings with karyotic membranes. PPIs have regulatory effects on various cellular processes, but their roles in plants are currently not PtdIns(3,5)P2, corroborating that this PPI is important for vacuole morphology. Taken together, these results provide evidence that well-understood. Plant growth relies largely on turgor-driven PPIs, together with their metabolic enzymes SAC2–SAC5, are cru- cell expansion, which at the subcellular level is linked to vacu- cial for vacuolar trafficking and for vacuolar morphology and func- olar dynamics. We identified an unknown subgroup of tonoplast- Arabidopsis thaliana tion in plants. associated enzymes from , the suppressor of actin 2 (SAC2) to SAC5, that modify PPI levels in plants and in- Arabidopsis membrane lipids | epidermal cells | membrane fusion and fission fluence vacuolar morphology. lines overexpressing or deficient in SAC isoforms display growth aberrations consistent with defective vacuolar function and turgor control. The data hint olyphosphoinositides (PPIs) are a class of signaling mem- at PPI-regulated processes in the plant tonoplast and link PPIs to Pbrane lipids, comprising the phosphorylated products of the control of turgor-driven cell expansion and, possibly, other phosphatidylinositol (PtdIns). PPIs perform a dual function as vacuolar functions. scaffolding signals and precursors for other molecular mes- sengers, which, together with their specific distribution at dif- Author contributions: P.N., S.H., E.F., R.T., M.V.M., I.H., T.M., and J.F. designed research; ferent intracellular membranes, makes PPIs important mediators P.N., S.H., E.F., R.T., R.v.W., T.V., M.H., J.L., R.D.R., M.I.F., and P.G. performed research; of a wide variety of cellular processes, such as membrane traf- P.N., S.H., E.F., R.T., R.v.W., M.H., I.H., T.M., and J.F. analyzed data; and P.N., S.H., R.T., T.V., ficking and homeostasis, cytoskeleton organization, nuclear sig- I.H., T.M., and J.F. wrote the paper. naling, and stress responses (1–5). The metabolism of PPIs is The authors declare no conflict of interest. regulated by specific kinases, phosphatases, and phospholipases Data deposition: The sequences reported in this paper have been deposited in the to tightly control the concentration and intracellular localization GenBank database [accession nos. AAP49834.1 (At1G22620, SAC1), AAP49835.1 (At3G14205, SAC2), AAP49836.1 (At3G43220, SAC3), AAP49837.1 (At5G20840, SAC4), AAP49838.1 of different lipid pools (2, 6, 7). (At1G17340, SAC5), and AAC49823.1 (At5G46860, SYP22)]. In yeast, two phosphoinositide (PI) types, PtdIns3P and PtdIns 1Present address: Institute of Plant Biology, University of Zürich, CH-8008 Zürich, (3,5)P2, and their interconversion have been shown to play cru- Switzerland. cial roles in trafficking toward the vacuole, regulation of vacuolar 2 – Present address: Department of Applied Genetics and Cell Biology, University of Natural pH, and vacuolar membrane fusion and fission (8 11). In yeast Resources and Life Sciences (BOKU), 1190 Vienna, Austria. and mammals, production and degradation of PtdIns(3,5)P2 in- 3Present address: Plant Molecular Biology Laboratory, Department of Biology, Faculty of volve the PtdIns3P 5-kinase Fab1p/PIKfyve and the antagonistic Sciences, University of Chile, Santiago 7800003, Chile. phosphatase factor-induced gene/suppressor of actin 3 (Fig4/ 4To whom correspondence may be addressed. E-mail: [email protected] or jiri. Sac3), respectively (8, 12–14). Impairment of genes implicated in [email protected]. PtdIns(3,5)P2 metabolism has deleterious consequences in yeast, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plants, and mammals (8, 15–19), demonstrating an essential 1073/pnas.1324264111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1324264111 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 with PtdIns3P to the tonoplast and maintain the morphology of storage and lytic vacuoles. Our results demonstrate the crucial role of PPIs and SAC domain phosphatases in the function and morphology of vacuoles in plants. Results and Discussion Evolution of SAC Phosphatases in the Green Lineage. We recon- structed the evolution of SAC domain-containing proteins in the green lineage (Fig. S1) based on full-length protein sequences. Similar to previous reports (26), we identified three major clades containing fungal, algal, and land plant sequences that probably originated before or early during eukaryote diversification. The first clade contains Arabidopsis SAC1–SAC5, which is most ho- mologous to the yeast Fig4p protein, but with SAC1 distinct from the SAC2–SAC5 subgroup that probably originated after land plant diversification and, subsequently, led to multiple copies in Arabidopsis. The second clade consists of SAC6–SAC8, homol- ogous to the yeast Sac1p protein that plays a role in the main- tenance of PtdIns4P levels and to the inositol polyphosphate 5-phosphatases INP51–INP53 that are also known as the syn- aptojanin-like proteins SJL1, SJL2, and SJL3 (27, 28). The third clade contains SAC9, the longest of the SAC domain-containing proteins from Arabidopsis, but with no yeast homolog, although homologs can be identified in other fungal species. We focused on the first clade, particularly on the functionally uncharac- terized SAC2–SAC5 subgroup that diverged in land plants and is seemingly distinct from the previously characterized SAC1 (25). SAC2–SAC5 Phosphatases Localize to the Tonoplast. The closest ortholog of SAC1–SAC5 in yeast is Fig4p, which exhibits a phosphatase activity against PtdIns(3,5)P2 and is localized to the tonoplast of the central vacuole (12, 13). In vitro, Arabidopsis SAC1 also exhibits phosphatase activity toward PtdIns(3,5)P2, but localizes to the Golgi apparatus (25). To gain insights into the subcellular localization of SAC2–SAC5, we generated trans- genic Arabidopsis lines expressing SAC genes fused to green fluorescent protein (GFP) or red fluorescent protein (RFP) under their native or 35S constitutive promoters. In root epi- 35S::SAC2-GFP 35S::SAC5- dermal cells of plants expressing to – – GFP Fig. 1. Localization of SAC2 SAC5 to the tonoplast. (A C) Fluorescence , the fluorescent signal was detected mostly at the tonoplast, microscopy of SAC2-GFP and SAC5-GFP, which localize to the tonoplast un- with faint fluorescence in the cytoplasm (Fig. 1A and Fig. S2 A– der the 35S (A) and native promoters (B and C). (D–F) Colocalization of C). SAC2-GFP and SAC5-GFP expression under the endoge- SYP22-YFP with 35S::SAC5-RFP (D) and SAC5::SAC5-GFP with the endocytic nous promoters localized only in the tonoplast (Fig. 1 B and C).
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