SAC phosphoinositide phosphatases at the tonoplast mediate vacuolar function in Arabidopsis

Abstract

Phosphatidylinositol (PtdIns) is a structural phospholipid that can be phosphorylated into various lipid signaling molecules, designated polyphosphoinositides (PPIs). The reversible phosphorylation of PPIs on the 3, 4, or 5 position of inositol is performed by a set of organelle-specific kinases and phosphatases, and the characteristic head groups make these molecules ideal for regulating biological processes in time and space. In yeast and mammals, PtdIns3P and PtdIns(3,5)P 2 play crucial roles in trafficking toward the lytic compartments, whereas the role in plants is not yet fully understood. Here we identified the role of a land plant-specific subgroup of PPI phosphatases, the suppressor of actin 2 (SAC2) to SAC5, during vacuolar trafficking and morphogenesis in Arabidopsis thaliana. SAC2-SAC5 localize to the tonoplast along with PtdIns3P, the presumable product of their activity. In SAC gain-and loss-of-function mutants, the levels of PtdIns monophosphates and bisphosphates were changed, with opposite effects on the morphology of storage and lytic vacuoles, and the trafficking toward the vacuoles was defective. Moreover, multiple 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 developmental defects. The fragmented vacuolar phenotype of sac mutants could be mimicked by treating wild-type seedlings with PtdIns(3,5)P 2 , corroborating that this PPI is important for vacuole morphology. Taken together, these results provide evidence that PPIs, together with their metabolic enzymes SAC2-SAC5, are crucial for vacuolar trafficking and for vacuolar morphology and function in plants.membrane lipids | epidermal cells | membrane fusion and fission P olyphosphoinositides (PPIs) are a class of signaling membrane lipids, comprising the phosphorylated products of phosphatidylinositol (PtdIns). PPIs perform a dual function as scaffolding signals and precursors for other molecular messengers, which, together with their specific distribution at different intracellular membranes, makes PPIs important mediators of a wide variety of cellular processes, such as membrane trafficking and homeostasis, cytoskeleton organization, nuclear signaling, and stress responses (1-5). The metabolism of PPIs is regulated by specific kinases, phosphatases, and phospholipases to tightly control the concentration and intracellular localization of different lipid pools (2, 6, 7).In yeast, two phosphoinositide (PI) types, PtdIns3P and PtdIns (3,5)P 2 , and their interconversion have been shown to play crucial roles in trafficking toward the vacuole, regulation of vacuolar pH, and vacuolar membrane fusion and fission (8-11). In yeast and mammals, production and degradation of PtdIns(3,5)P 2 involve the PtdIns3P 5-kinase Fab1p/PIKfyve and the antagonistic phosphatase factor-induced gene/suppressor of actin 3 (Fig4/ Sac3), respectively (8,(12)(13)(14). Impairment of genes implicated in PtdIns(3,5)P 2 metabolism ...

Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses

“…In turn, genetic evidence indicates that PtdIns(3,5)P 2 might be produced at the tonoplast and late endosomes (Heilmann and Heilmann, 2015;Hirano et al, 2015;Novakova et al, 2014;Simon et al, 2014Simon et al, , 2016Vermeer et al, 2006Vermeer et al, , 2009. The cellular distribution of PtdIns5P in plants is unknown and neither PtdIns (3,4)P 2 nor PtdIns(3,4,5)P 3 have as yet been detected in plants (Heilmann, 2009;Munnik and Nielsen, 2011).…”

Phosphoinositide signaling in plant development

“…In recent years, PIs have emerged as central regulators of plant function and development, and a number of important and severe plant phenotypes have been reported when plant PI metabolism is perturbed Nelson, 2009, 2004;Chen et al, 2008;Ercetin et al, 2008;Golani et al, 2013;Gunesekera et al, 2007;Ischebeck et al, 2008Ischebeck et al, , 2011Ischebeck et al, , 2013Kaye et al, 2011;Kusano et al, 2008;Löfke et al, 2008;Mei et al, 2012;Novakova et al, 2014;Parker et al, 2000;Preuss et al, 2006;Rodriguez-Villalon et al, 2015;Sousa et al, 2008;Stenzel et al, 2008;Tejos et al, 2014;Wang et al, 2012;Williams et al, 2005;Zhong et al, , 2005. The perturbation of PI signaling components in Arabidopsis, for example, gives rise to a variety of phenotypes affecting multiple tissue types and aspects of plant development (summarized in Table 1).…”

“…For instance, the perturbation of PIP5K2 (Stenzel et al, 2012) or PIP5K10 and PIP5K11 (Ischebeck et al, 2011) results in altered actin dynamics and the loss of cellular polarity. Yet a different category of phenotypes indicates that 3-phosphorylated PIs, which are perturbed by altered levels of VPS34 (Lee et al, 2008a,b), Fab1-like proteins (Bak et al, 2013) or SAC2-SAC5 (Novakova et al, 2014), might be involved in endomembrane trafficking and the control of vacuolar functions. The overall scheme suggests that PIs are involved in controlling the central machinery for membrane trafficking and protein sorting in plants.…”

Actin-dependent vacuolar occupancy of the cell determines auxin-induced growth repression

“…Auxin treatment seemingly leads to the formation of multiple small luminal vacuoles (15), and several studies previously have addressed the mechanisms of vacuolar fragmentation (16,18,(23)(24)(25). Accordingly, actin and its motor protein myosin could contribute the force required for auxin-dependent vacuolar fission and fusion events.…”