Phosphatidylinositol 3-Phosphate 5-Kinase,FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis

Phosphatidylinositol 3,5-bisphosphate PtdIns(3,5)P₂] is an important lipid in membrane trafficking in animal and yeast systems; however, its role is still largely obscure in plants. Here, we demonstrate that the phosphatidylinositol 3-phosphate 5-kinase, formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), and its product, PtdIns(3,5)P₂, are essential for the maturation process of endosomes to mediate cortical microtubule association of endosomes, thereby controlling proper PIN-FORMED protein trafficking in young cortical and stele cells of root. We found that FAB1 predominantly localizes on the Sorting Nexin1 (SNX1)-residing late endosomes, and a loss of FAB1 function causes the release of late endosomal proteins, Ara7, and SNX1 from the endosome membrane, indicating that FAB1, or its product PtdIns(3,5)P₂, mediates the maturation process of the late endosomes. We also found that loss of FAB1 function causes the release of endosomes from cortical microtubules and disturbs proper cortical microtubule organization.Phosphoinositides play an important role in various cellular processes, including determination of organelle identity and mediating signal transduction by recruiting effector molecules to various organelles (Balla, 2013). Among those, D3-phosphorylated phosphoinositides, phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate PtdIns(3,5)P₂], play essential roles in the endosomal trafficking and the vacuolar sorting. PtdIns3P is produced from phosphatidylinositol by class III PI3-kinase, vacuolar protein sorting34 (VPS34). In animal cells, PtdIns3P predominantly localizes to the early endosomes and controls endosome maturation, recycling, and degradation of cargo proteins coordinated with Rab5 GTPases (Jean and Kiger, 2012). In Arabidopsis (Arabidopsis thaliana), PtdIns3P mainly resides on the late endosomes and the prevacuolar membrane (Vermeer et al., 2006; Simon et al., 2014). Dysfunction of AtVPS34 resulted in a defect in growth (Welters et al., 1994), root hair elongation (Lee et al., 2008a), and pollen development (Lee et al., 2008b), indicating an important role for AtVPS34 and its product PtdIns3P in plant development. VPS34-mediated PtdIns3P synthesis at the endosomes recruits phosphatidylinositol 3-phosphate 5-kinase formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), then FAB1/PIKfyve produces PtdIns(3,5)P₂ from PtdIns3P to mediate late endosome maturation in yeast (Saccharomyces cerevisiae) and animals (Ho et al., 2012; Jean and Kiger, 2012). PtdIns(3,5)P₂ has crucial roles in the maintenance of lysosome/vacuole morphology and acidification, membrane trafficking of proteins, autophagy, and signaling mediation in response to various stresses (Shisheva, 2008).FAB1 was discovered in yeast, where mutations were found to result in the formation of aploid and binucleate cells (hence its name FAB). In addition, a loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth (Yamamoto et al., 1995). In mammalian cells, this kinase is called PIKfyve (FYVE is a PI3P-binding domain). FAB1/PIKfyve forms a protein complex with an adaptor-like protein, Vacuole14 (Bonangelino et al., 1997) and PtdIns(3,5)P₂ 5-phosphatase (Fig. 4; Gary et al., 2002), indicating that the FAB1 complex catalyzes both PtdIns(3,5)P₂ synthesis and turnover simultaneously. In mammalian cells, interference of FAB1/PIKfyve function causes severe defects during embryogenesis, resulting in embryonic lethality in Drosophila spp., Caenorhabditis elegans, and mice (Nicot et al., 2006; Rusten et al., 2006; Ikonomov et al., 2011; Takasuga et al., 2013). Whereas most genomes from human to yeast contain a single-copy gene, the Arabidopsis genome codes for four FAB1 genes (FAB1A–D), of which only FAB1A and FAB1B contain a FYVE domain (Mueller-Roeber and Pical, 2002), and fab1a/fab1b double mutant reveals male gametophyte lethality phenotype in Arabidopsis (Whitley et al., 2009). The mutant pollen shows severe defects in vacuolar reorganization following the first mitotic division of development, suggesting an important role of FAB1 and PtdIns(3,5)P₂ in vacuolar rearrangement for pollen development (Whitley et al., 2009).Open in a separate windowFigure 4.Localization of endosomal markers upon down-regulation of FAB1A/B or inhibition of PtdIns(3,5)P₂ synthesis in young root cortical cells. Localization of mRFP-SYP43, mRFP-vesicle-associated membrane protein (VAMP727), mRFP-ARA7, and SNX1-mRFP without estradiol (A, E, I, and M) or with estradiol (B, F, J, and N) in the FAB1A/B-amiRNA line, or wild-type (WT) plants without YM201636 (C, G, K, and O) or with YM201636 (D, H, L, and P). Bar = 10 μm. Measurement of fluorescent dot structures (Q). Data represent fluorescent dots per cell (mean ± sd). *, P < 0.001 (Student’s t test).We previously developed a transgenic Arabidopsis line that is able to conditionally down-regulate FAB1A and FAB1B expression simultaneously, and demonstrated that a loss of FAB1 function causes various abnormal phenotypes, including growth inhibition, hypersensitivity to exogenous auxin, disturbance of root gravitropism, and floral organ abnormalities (Hirano et al., 2011). In addition, we found that down-regulation of FAB1A/B expression impaired endomembrane homeostasis, including endocytosis, vacuole formation, and vacuolar acidification, likely causing pleiotropic developmental phenotypes that mostly related to the auxin signaling in Arabidopsis (Hirano et al., 2011; Hirano and Sato, 2011). In plants, auxin is a crucial phytohormone that has a wide variety of physiological roles associated with growth, development, and tropic responses (Zhao, 2010). The polar cell-to-cell transport of auxin is mediated by auxin transporters localized on the plasma membrane (PM), such as PIN-FORMED (PIN) proteins (Vieten et al., 2007; Feraru and Friml, 2008). PINs are used as model molecules for polarity establishment on the PM in Arabidopsis. The establishment of PIN polarity is accomplished by the recycling of PINs between the PM and endosomal compartments comprising the trans-Golgi network/early endosomes (TGN/EEs) and the late endosomes (LEs)/prevacuolar compartments. The PIN-recycling pathway is mediated by multiple endosomal regulatory proteins, such as Rab family GTPases and Sorting Nexin (SNX; Jaillais et al., 2006; Park and Jürgens, 2011).Rab proteins function as molecular switches to regulate the tethering and fusion step of transport vesicles to target membranes. Rab5 members of the Rab GTPases have various functions in the endocytic pathway in eukaryotes. The maturation of the early-to-late endosomes is regulated by Rab5-to-Rab7 conversion, which is regulated by the Mon1/Sand-1-Ccz1 complex (Nordmann et al., 2010; Poteryaev et al., 2010). In plants, Rab5-family proteins, Ara6 and Ara7, and Rha1 play important roles in Rab5-mediated endosomal trafficking including the vacuolar trafficking pathway, thereby regulating of the polar transport of auxin and responses to environmental conditions (Ebine et al., 2011; Inoue et al., 2013).SNXs are composed of two conserved domains: the PHOX domain, involved in the interaction with the phosphoinositides, PtdIns3P and PtdIns (3,5)P₂, in the endosomal membrane in animals (Cozier et al., 2002), and the BAR domain, mediating dimerization and binding to curved membranes (Peter et al., 2004). Loss of SNX function disrupts the stable association of the retromer subcomplex, VPS26-Vps29-Vps35, with endosomal membranes, and thus results in retromer dysfunction, indicating that SNXs have a crucial role in the assembly and maintenance of the core retromer function (Teasdale et al., 2001; Cullen and Korswagen, 2012). The first plant SNX was identified as a protein that interacts with various receptor kinases in Brassica oleracea (Vanoosthuyse et al., 2003), and then three SNX genes (SNX1, SNX2a, and SNX2b) were identified in Arabidopsis. The snx1 null mutant exhibits a semidwarf phenotype with other subtle developmental defects (Pourcher et al., 2010). SNX1 is localized to the late endosome and is involved in PIN2 recycling between endosomes and the PM (Jaillais et al., 2006). SNX1 has been reported to interact with cortical microtubules via the microtubule-associated protein Cytoplasmic Linker Associated Protein (CLASP), and the clasp1 null mutant displays aberrant SNX1 endosomes and enhanced PIN2 degradation in the lytic vacuoles, suggesting that an association of SNX1 endosomes and CLASP is important for recycling of PIN transporters (Ambrose et al., 2013).Although many analyses of FAB1/PIKfyve, Rab5 family GTPases, SNXs, and microtubles have been reported, and there are significant similarities in endosomal trafficking, a functional relationship between them is still largely obscure.In this study, we demonstrate that FAB1 produced PtdIns(3,5)P₂ in Arabidopsis, and knockdown of FAB1 expression or inhibition of FAB1 activity with a FAB1/PIKfyve inhibitor, YM201636, decreased PtdIns(3,5)P₂ content. We also found that FAB1 and its product PtdIns(3,5)P₂ mediate the late endosome maturation by recruiting endosomal effector molecules, Ara7 and SNX1, onto endosomes to establish endosome-cortical microtubule interaction. Subsequently, the basal polarity of PIN2 in young cortical cells and PIN1 in stele cells is achieved.