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 ...

VSTx1 is a voltage sensor toxin from the spider Grammostola spatulata that inhibits KvAP, an archeabacterial voltage-activated K(+) channel whose X-ray structure has been reported. Although the receptor for VSTx1 and the mechanism of inhibition are unknown, the sequence of the toxin is related to hanatoxin (HaTx) and SGTx, two toxins that inhibit eukaryotic voltage-activated K(+) channels by binding to voltage sensors. VSTx1 has been recently shown to interact equally well with lipid membranes that contain zwitterionic or acidic phospholipids, and it has been proposed that the toxin receptor is located within a region of the channel that is submerged in the membrane. As a first step toward understanding the inhibitory mechanism of VSTx1, we determined the three-dimensional solution structure of the toxin using NMR. Although the structure of VSTx1 is similar to HaTx and SGTx in terms of molecular fold and amphipathic character, the detailed positions of hydrophobic and surrounding charged residues in VSTx1 are very different than what is seen in the other toxins. The amphipathic character of VSTx1, notably the close apposition of basic and hydrophobic residues on one face of the toxin, raises the possibility that the toxin interacts with interfacial regions of the membrane. We reinvestigated the partitioning of VSTx1 into lipid membranes and find that VSTx1 partitioning requires negatively charged phospholipids. Intrinsic tryptophan fluorescence and acrylamide quenching experiments suggest that tryptophan residues on the hydrophobic surface of VSTx1 have a diminished exposure to water when the toxin interacts with membranes. The present results suggest that if membrane partitioning is involved in the mechanism by which VSTx1 inhibits voltage-activated K(+) channels, then binding of the toxin to the channel would likely occur at the interface between the polar headgroups and the hydrophobic phase of the membrane.

Deformed wing virus (DWV) is a serious pathogen of the honey bee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In the present study we reduced DWV infection by feeding both first instar larvae and adult A. mellifera with a double-stranded (ds) RNA construct, DWV-dsRNA, which is specific to DWV in DWV-inoculated bees, by mixing it with their food. We showed that feeding DWV to larvae causes wing deformity in adult bees in the absence of varroa mites and decreases survival rates of adult bees relative to bees not fed DWV. Feeding larvae with DWV-dsRNA in advance of inoculation with virus reduced the DWV viral level and reduced wing deformity relative to larvae fed DWV or DWV with green fluorescent protein-dsRNA (probably a result of RNA silencing), but did not affect survival to the adult stage. Feeding DWV-dsRNA did not affect larval survival rates, which suggests that dsRNA is non-toxic to larvae. Feeding adult workers with DWV-dsRNA in advance of inoculation with virus increased their longevity and reduced DWV concentration relative to controls.

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 trans-Golgi 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)P-dependent 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.

An efficient cell transformation method is presented that utilizes droplet electroporation on a microfluidic chip. Two types of green microalgae, a wall-less mutant and a wild type of Chlamydomonas reinhardtii, are used as model cells. The PDMS-glass electroporation chip is simply composed of a flow-focusing microstructure for generating cell-encapsulating droplets and a serpentine channel for better mixing of the content in the droplet, and five pairs of parallel microelectrodes on the glass slide, without involving any expensive electrical equipment. The transformation efficiency via the microfluidic electroporation is shown to be more than three orders of magnitude higher for the wall-less mutant, and more than two orders of magnitude higher for the wild type, which has its cell wall intact, than bulk phase electroporation under identical conditions. Furthermore, the microfluidic transformation is remarkably efficient even at a low DNA/cell ratio, facilitating ways of controlling the transgenic copy number, which is important for the stability of the transgene expression.

Antimicrobial peptides are produced as components of the innate immune system by a wide variety of insects, amphibians, and mammals, including humans [1][2][3][4]. In recent decades, the structures and functions of many antimicrobial peptides have been extensively studied to elucidate their mode of action. Typically, antimicrobial

Amphipathic protein toxins from tarantula venom inhibit voltage-activated potassium (Kv) channels by binding to a critical helix-turn-helix motif termed the voltage sensor paddle. Although these toxins partition into membranes to bind the paddle motif, their structure and orientation within the membrane are unknown. We investigated the interaction of a tarantula toxin named SGTx with membranes using both fluorescence and NMR spectroscopy. Depth-dependent fluorescence-quenching experiments with brominated lipids suggest that Trp30 in SGTx is positioned approximately 9 A from the center of the bilayer. NMR spectra reveal that the inhibitor cystine knot structure of the toxin does not radically change upon membrane partitioning. Transferred cross-saturation NMR experiments indicate that the toxin's hydrophobic protrusion contacts the hydrophobic core of the membrane, whereas most surrounding polar residues remain at interfacial regions of the bilayer. The inferred orientation of the toxin reveals a twofold symmetry in the arrangement of basic and hydrophobic residues, a feature that is conserved among tarantula toxins. These results have important implications for regions of the toxin involved in recognizing membranes and voltage-sensor paddles, and for the mechanisms by which tarantula toxins alter the activity of different types of ion channels.

Arabidopsis FY, a homologue of the yeast RNA 3' processing factor Pfs2p, regulates the autonomous floral transition pathway through its interaction with FCA, an RNA binding protein. It is demonstrated here that FY also influences seed dormancy. Freshly-harvested seed of the Arabidopsis fy-1 mutant germinated readily in the absence of stratification or after-ripening. Furthermore, the fy-1 mutant showed less ABA sensitivity compared with the wild type, Ler, under identical conditions. Freshly-harvested seed of fy-1 had significantly higher ABA levels than Ler, even though Ler was dormant and fy-1 germinated readily. The PPLPP domains of FY, which are required for flowering control, were not essential for the ABA-influenced repression of germination. FLC expression analysis in seeds of different genotypes suggested that the effect of FY on dormancy may not be elicited through FLC. No significant differences in CYP707A1, CYP707A2, NCED9, ABI3, and ABI4 were observed between freshly-harvested Ler and fy-1 imbibed for 48 h. GA3ox1 and GA3ox2 rapidly increased over the 48 h imbibition period for fy-1, with no significant increases in these transcripts for Ler. ABI5 levels were significantly lower in fy-1 over the 48 h imbibition period. The results suggest that FY is involved in the development of dormancy and ABA sensitivity in Arabidopsis seed.