Trafficking of Phosphatidylinositol 3-Phosphate from the trans-Golgi Network to the Lumen of the Central Vacuole in Plant Cells

Abstract

Very limited information is available on the role of phosphatidylinositol 3-phosphate (PI[3]P) in vesicle trafficking in plant cells. To investigate the role of PI(3)P during the vesicle trafficking in plant cells, we exploited the PI(3)P-specific binding property of the endosome binding domain (EBD) (amino acids 1257 to 1411) of human early endosome antigen 1, which is involved in endosome fusion. When expressed transiently in Arabidopsis protoplasts, a green fluorescent protein (GFP):EBD fusion protein exhibited PI(3)P-dependent localization to various compartments-such as the transGolgi network, the prevacuolar compartment, the tonoplasts, and the vesicles in the vacuolar lumen-that varied with time. The internalized GFP:EBD eventually disappeared from the lumen. Deletion experiments revealed that the PI(3)Pdependent localization required the Rab5 binding motif in addition to the zinc finger motif. Overexpression of GFP:EBD inhibited vacuolar trafficking of sporamin but not trafficking of H ؉ -ATPase to the plasma membrane. On the basis of these results, we propose that the trafficking of GFP:EBD reflects that of PI(3)P and that PI(3)P synthesized at the trans -Golgi network is transported to the vacuole through the prevacuolar compartment for degradation in plant cells. INTRODUCTIONEvidence suggests that phosphoinositides play a regulatory role in vesicle trafficking (Camilli et al., 1996; Corvera and Czech, 1998; Gary et al., 1998; Cremona et al., 1999;Leevers et al., 1999;Roth, 1999). Phosphatidylinositol 3-phosphate (PI[3]P) has been implicated in this process (Whitman et al., 1998; Corvera et al., 1999). Direct evidence for the role of PI(3)P in vesicle trafficking was obtained when the yeast VPS34 gene, one of the genes involved in the vesicular protein sorting in yeast, was found to encode a PI3-kinase (Schu et al., 1993). Almost all PI3-kinase activity in yeast can be attributed to Vps34p (Stack et al., 1995;Wurmser and Emr, 1998). However, Vps34p requires a protein kinase, Vps15p, for its activation and membrane association (Stack et al., 1995). Also, a mammalian protein, p110, which is homologous to yeast Vps34p, has PI3-kinase activity and can transduce signals from tyrosine-phosphorylated receptors into a variety of intracellular responses (Volinia et al., 1995). However, this mammalian PI3-kinase, in association with an adapter protein p85, is able to phosphorylate other PI compounds such as PI(4)P and PI(4,5)P 2 at the D3 position of PI (Volinia et al., 1995;Panaretou et al., 1997). It is now clear that PI3-kinases play critical roles in various trafficking events, such as endocytosis of transferrin (Li et al., 1995), endosome fusion (Jones et al., 1998), vacuolar trafficking in yeast (Peterson et al., 1999), vesicle formation at the transGolgi network (TGN) (Hickinson et al., 1997; Jones and Howell, 1997; Jones et al., 1998), vacuole morphogenesis in Schizosaccharomyces pombe (Takegawa et al., 1995), and multivesicular body formation (Fernandez-Borja et al., 1999).PI(3)P is likely to act in ...

The Arabidopsis Phosphatidylinositol 3-Kinase Is Important for Pollen Development

“…In yeast, protein transport to the vacuole depends on the Vam7 t-SNARE, and its targeting to the vacuole is dependent on PtdIns(3)P generation (Cheever et al, 2001). The role of PtdIns(3)P in vacuolar sorting has also been reported in plant cells (Matsuoka et al, 1995;Kim et al, 2001;Tse et al, 2004;daSilva et al, 2005;Oliviusson et al, 2006). Therefore, shortage of PtdIns(3)P may lead to failure in the recruitment and targeting of proteins necessary for vacuolar function, thus causing defects in vacuolar dynamics.…”

“…Since PtdIns(3)P is important for trafficking of vacuolar proteins in somatic plant cells (Matsuoka et al, 1995;Kim et al, 2001;Oliviusson et al, 2006), and because dynamic changes in vacuolar morphology is a prominent feature during pollen development (Yamamoto et al, 2003), we were curious whether the Table I. Segregation analysis of VPS34/vps34-1 and VPS34/vps34-2…”

“…In animal cells, inhibition of PtdIns(3)P synthesis by pharmacological agents impairs the targeting of procathepsin D from the trans-Golgi network to the lysosomal compartment (Brown et al, 1995;Davidson, 1995). In plants as well, overexpression of PtdIns(3)Pbinding protein in Arabidopsis (Arabidopsis thaliana) protoplasts inhibits trafficking of the vacuolar protein sporamin (Kim et al, 2001). A similar failure to deliver vacuolar proteins in tobacco (Nicotiana tabacum) suspension cells treated with pharmacological agents that interfere with PtdIns(3)P synthesis was reported (Matsuoka et al, 1995).…”

Auxin‐induced reactive oxygen species production requires the activation of phosphatidylinositol 3‐kinase

“…Although type III PtdIns 3-kinase has also been identified in plant cells, its role has yet to be determined. It has been suggested that PtdIns(3)P is involved in intracellular vesicle trafficking by binding to the FYVE domain [11,21]. Bunney et al demonstrated that PtdIns 3-kinase is associated with active nuclear or nucleolar transcription sites, suggesting a role in the regulating transcription [27].…”

“…Arabidopsis protoplasts were isolated with an enzyme mixture containing 0.25% macerozyme R-10, 400 mM mannitol, 1 mM CaCl 2 and 5 mM MES-KOH (pH 5.6) and transfected by a polyethylene glycol-based method as previously described [21]. The viability of protoplasts was determined by staining with 1% Evans Blue.…”