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 ...

Epsin and related proteins play important roles in various steps of protein trafficking in animal and yeast cells. Many epsin homologs have been identified in plant cells from analysis of genome sequences. However, their roles have not been elucidated. Here, we investigate the expression, localization, and biological role in protein trafficking of an epsin homolog, Arabidopsis thaliana EPSIN1, which is expressed in most tissues we examined. In the cell, one pool of EPSIN1 is associated with actin filaments, producing a network pattern, and a second pool localizes primarily to the Golgi complex with a minor portion to the prevacuolar compartment, producing a punctate staining pattern. Protein pull-down and coimmunoprecipitation experiments reveal that Arabidopsis EPSIN1 interacts with clathrin, VTI11, γ-adaptin-related protein (γ-ADR), and vacuolar sorting receptor1 (VSR1). In addition, EPSIN1 colocalizes with clathrin and VTI11. The epsin1 mutant, which has a T-DNA insertion in EPSIN1, displays a defect in the vacuolar trafficking of sporamin:green fluorescent protein (GFP), but not in the secretion of invertase:GFP into the medium. Stably expressed HA:EPSIN1 complements this trafficking defect. Based on these data, we propose that EPSIN1 plays an important role in the vacuolar trafficking of soluble proteins at the trans-Golgi network via its interaction with γ-ADR, VTI11, VSR1, and clathrin.

Plant development is regulated by numerous chemicals derived from a multitude of metabolic pathways. However, we know very little about the biological effects and functions of many of these metabolites in the cell. N-Acylethanolamines (NAEs) are a group of lipid mediators that play important roles in mammalian physiology. Despite the intriguing similarities between animals and plants in NAE metabolism and perception, not much is known about the precise function of these metabolites in plant physiology. In plants, NAEs have been shown to inhibit phospholipase Dalpha (PLDalpha) activity, interfere with abscisic acid-induced stomatal closure, and retard Arabidopsis seedling development. 1-Butanol, an antagonist of PLD-dependent phosphatidic acid production, was reported to induce defects in Arabidopsis seedling development that were somewhat similar to effects induced by elevated levels of NAE. This raised the possibility that the impact of NAE on seedling growth could be mediated in part via its influence on PLD activity. To begin to address this possibility, we conducted a detailed, comparative analysis of the effects of 1-butanol and N-lauroylethanolamine (NAE 12:0) on Arabidopsis root cell division, in vivo cytoskeletal organization, seed germination, and seedling growth. Although both NAE 12:0 and 1-butanol induced profound cytoskeletal and morphological alterations in seedlings, there were distinct differences in their overall effects. 1-Butanol induced more pronounced modifications in cytoskeletal organization, seedling growth, and cell division at concentrations severalfold higher than NAE 12:0. We propose that these compounds mediate their differential effects on cellular organization and seedling growth, in part through the differential modulation of specific PLD isoforms.

LONG HYPOCOTYL5 (HY5) is a bZIP (basic leucine zipper) transcription factor that activates photomorphogenesis and root development in Arabidopsis (Arabidopsis thaliana). Previously, STF1 (soybean [Glycine max] TGACG-motif binding factor 1), a homologous legume protein with a RING-finger motif and a bZIP domain, was reported in soybean. To investigate the role of STF1, the phenotypes of transgenic Arabidopsis plants overexpressing STF1 and HY5 were compared. In addition, the DNA-binding properties of STF1 and HY5 were extensively studied using random binding site selection and electrophoretic mobility shift assay. Overexpression of STF1 in the hy5 mutant of Arabidopsis restored wild-type photomorphogenic and root development phenotypes of short hypocotyl, accumulation of chlorophyll, and root gravitropism with partial restoration of anthocyanin accumulation. This supports that STF1 is a homolog of HY5 with a role in light and hormone signaling. The DNA-binding properties of STF1 and HY5 are shown to be similar to each other in recognizing many ACGT-containing elements with a consensus sequence motif of 5#-( G / A )( G / A ) TGACGT( C /G/ A )( A /T/ G )-3#. The motif represents a characteristically strong preference for flanking sequence to TGACGT and a larger sequence than the sequences recognized by the G-box binding factor and TGA protein families. The finding of C-box, hybrid C/G-, and C/A-boxes as high-affinity binding sites over the G-box and parameters associated with HY5 recognition define the criteria of HY5/STF1 protein-DNA interaction in the promoter regions. This study helps to predict the precise in vivo binding sites of the HY5 protein from the vast number of putative HY5 genomic binding sites analyzed by chromatin immunoprecipitation on chip.