Protein transport to the yeast vacuole.

Genetic and biochemical analyses of yeast vacuolar protein localization have identified more than 40 gene products that play a role in this process. Included among these components are a sorting receptor, a protein kinase, a phosphatidylinositol kinase, small GTP-binding proteins and a dynamin-like GTPase. Some of these gene products are homologous to proteins required for sorting and transport at other stages of the secretory and endocytic pathways. Others appear to be required for unique functions in the vacuolar protein localization pathway. Recent studies have helped to define the role that each of these components plays in vacuolar protein localization and have offered new insights into the molecular mechanisms of protein sorting.

Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells.

Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role for a phosphatidylinositol-specific PI 3-kinase, the product of the VPS34 gene, in the targeting of newly synthesized proteins to the vacuole, an organelle functionally equivalent to mammalian lysosomes (Schu, P. V., K. Takegawa, M. J. Fry, J. H. Stack, M. D. Waterfield, and S. D. Emr. 1993. Science [Wash. DC]. 260:88-91). The activity of Vps34p kinase is significantly reduced by the PI 3-kinase inhibitors wortmannin, a fungal metabolite, and LY294002, a quercetin analog (Stack, J. H., and S. D. Emr. 1994. J. Biol. Chem. 269:31552-31562). We show here that at concentrations which inhibit VPS34-encoded PI 3-kinase activity, wortmannin also inhibits the processing and delivery of newly synthesized cathepsin D to lysosomes in mammalian cells with half-maximal inhibition of delivery occurring at 100 nM wortmannin. As a result of wortmannin action, newly synthesized, unprocessed cathepsin D is secreted into the media. Moreover, after accumulation in the trans-Golgi network (TGN) at 20 degrees C, cathepsin D was rapidly missorted to the secretory pathway after addition of wortmannin and shifting to 37 degrees C. At concentrations that inhibited lysosomal enzyme delivery, both wortmannin and LY294002 caused a highly specific dilation of mannose 6-phosphate receptor (M6PR)-enriched vesicles of the prelysosome compartment (PLC), which swelled to approximately 1 micron within 15 min after treatment. With increasing time, the inhibitors caused a significant yet reversible change in M6PR distribution. By 3 h of treatment, the swollen PLC vacuoles were essentially depleted of receptors and, in addition, there was a fourfold loss of receptors from the cell surface. However, M6PRs were still abundant in the TGN. These results are most consistent with the interpretation that PI 3-kinase regulates the trafficking of lysosomal enzymes by interfering with a M6PR-dependent sorting event in the TGN. Moreover, they provide evidence that trafficking of soluble hydrolases to mammalian lysosomes and yeast vacuoles rely on similar regulatory mechanisms.

Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast.

A membrane-associated complex composed of the Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase (PtdIns 3-kinase) is essential for the delivery of proteins to the yeast vacuole. An active Vps15p is required for the recruitment of Vps34p to the membrane and subsequent stimulation of Vps34p PtdIns 3-kinase activity. Consistent with this, mutations altering highly conserved residues in the lipid kinase domain of Vps34p lead to a dominant-negative phenotype resulting from titration of activating Vps15 proteins. In contrast, catalytically inactive Vps15p mutants do not produce a dominant mutant phenotype because they are unable to associate with Vps34p in a wild-type manner. These data indicate that an intact Vps15p protein kinase domain is necessary for the association with and activation of Vps34p, and they demonstrate that a functional Vps15p-Vps34p complex is absolutely required for the efficient delivery of proteins to the vacuole. Analysis of a temperature-conditional allele of VPS15, in which a shift to the nonpermissive temperature leads to a decrease in cellular PtdIns(3)P levels, indicates that the loss of Vps15p function leads to a defect in activation of Vps34p. In addition, characterization of a temperature-sensitive allele of VPS34 demonstrates that inactivation of Vps34p leads to the immediate missorting of soluble vacuolar proteins (e.g., carboxypeptidase Y) without an apparent defect in the sorting of the vacuolar membrane protein alkaline phosphatase. This rapid block in vacuolar protein sorting appears to be the result of loss of PtdIns 3-kinase activity since cellular PtdIns(3)P levels decrease dramatically in vps34 temperature-sensitive mutant cells that have been incubated at the nonpermissive temperature. Finally, analysis of the defects in cellular PtdIns(3)P levels in various vps15 and vsp34 mutant strains has led to additional insights into the importance of PtdIns(3)P intracellular localization, as well as the roles of Vps15p and Vps34p in vacuolar protein sorting.

Phosphate Modulates Transcription of Soybean VspB and Other Sugar-Inducible Genes.

The soybean vegetative storage protein genes VspA and VspB encode vacuolar glycoprotein acid phosphatases. Transcription of the Vsp is synergistically activated by jasmonic acid or methyl jasmonate (MeJA) and soluble sugars. The action of these modulators is mediated by two different DNA domains in the VspB promoter. In this study, we present new data regarding VspB regulation by sucrose and inorganic phosphate, which suggest a common mechanism of transcriptional control for Vsp and other sugar-inducible genes. We found that the sugar-mediated activation of VspB expression was inhibited by phosphate. Deletion analysis and transient assays in tobacco protoplasts identified a 130-bp DNA domain in the VspB promoter that mediates both sucrose induction and phosphate inhibition. Transcription mediated by this DNA domain was induced by phosphate elimination from the protoplast incubation medium, even in the absence of sucrose. The effect of sucrose and phosphate on VspB expression was studied in vivo in several ways. Depletion of phosphate from soybean cell cultures by the addition of mannose stimulated VspB expression, even in the absence of sucrose or MeJA. In illuminated soybean leaves treated with MeJA, inhibition of photosynthetic electron transport by DCMU decreased VspB expression. In contrast, VspB expression in soybean leaves stimulated by phosphate depletion was not influenced by DCMU. Moreover, sucrose-stimulated expression of the sugar-responsive genes lipoxygenase A and chalcone synthase of soybean and proteinase inhibitor II and class I patatin of potato was inhibited by phosphate. Like VspB, these genes were stimulated by phosphate depletion in the absence of exogenous sucrose. We propose that sugar-responsive genes are activated, in part, by accumulation of sugar-phosphates and concomitant reduction of cellular phosphate levels. These data may help explain recruitment of the Vsp, which encode acid phosphatases, as vegetative storage proteins.

The phosphatidylinositol 3-phosphate binding protein Vac1p interacts with a Rab GTPase and a Sec1p homologue to facilitate vesicle-mediated vacuolar protein sorting.

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein-protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.

A novel mechanism for localizing membrane proteins to yeast trans-Golgi network requires function of synaptojanin-like protein.

Localization of resident membrane proteins to the yeast trans-Golgi network (TGN) involves both their retrieval from a prevacuolar/endosomal compartment (PVC) and a "slow delivery" mechanism that inhibits their TGN-to-PVC transport. A screen for genes required for the slow delivery mechanism uncovered INP53, a gene encoding a phosphoinositide phosphatase. A retrieval-defective model TGN protein, A(F-->A)-ALP, was transported to the vacuole in inp53 mutants approximately threefold faster than in wild type. Inp53p appears to function in a process distinct from PVC retrieval because combining inp53 with mutations that block retrieval resulted in a much stronger phenotype than either mutation alone. In vps27 strains defective for both anterograde and retrograde transport out of the PVC, a loss of Inp53p function markedly accelerated the rate of transport of TGN residents A-ALP and Kex2p into the PVC. Inp53p function is cargo specific because a loss of Inp53p function had no effect on the rate of Vps10p transport to the PVC in vps27 cells. The rate of early secretory pathway transport appeared to be unaffected in inp53 mutants. Cell fractionation experiments suggested that Inp53p associates with Golgi or endosomal membranes. Taken together, these results suggest that a phosphoinositide signaling event regulates TGN-to-PVC transport of select cargo proteins.

Novel PI(4)P 5-kinase homologue, Fab1p, essential for normal vacuole function and morphology in yeast.

The FAB1 gene of budding yeast is predicted to encode a protein of 257 kDa that exhibits significant sequence homology to a human type II PI(4)P 5-kinase (PIP5K-II). The recently cloned human PIP5K-II specifically converts PI(4)P to PI(4,5)P2 (Boronenkov and Anderson, 1995). The region of highest similarity between Fab1p and PIP5K-II includes a predicted nucleotide binding motif, which is likely to correspond to the catalytic domain of the protein. Interestingly, neither PIP5K-II nor Fab1p exhibit significant homology with cloned PI 3-kinases or PI 4-kinases. fab1 mutations result in the formation of aploid and binucleate cells (hence the name FAB). In addition, loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth. Experiments with a temperature conditional fab1 mutant revealed that their vacuoles rapidly (within 30 min) enlarge to more than double the size upon shifting cells to the nonpermissive temperature. Additional experiments with the fab1 ts mutant together with results obtained with fab1 vps (vacuolar protein sorting defective) double mutants indicate that the nuclear division and cell surface integrity defects observed in fab1 mutants are secondary to the vacuole morphology defects. Based on these data, we propose that Fab1p is a PI(4)P 5-kinase and that the product of the Fab1p reaction, PIP2, functions as an important regulator of vacuole homeostasis perhaps by controlling membrane flux to and/or from the vacuole. Furthermore, a comparison of the phenotypes of fab1 mutants and other yeast mutants affecting PI metabolism suggests that phosphoinositides may serve as general regulators of several different membrane trafficking pathways.

Sucrose Modulation of Soybean Vsp Gene Expression Is Inhibited by Auxin.

We have shown that auxin represses soybean (Glycine max L.) vegetative storage protein gene (Vsp) expression in suspension-cultured cells and in leaves and petioles of excised trifoliates. The auxin analog naphthyleneacetic acid (NAA) at 10 [mu]M strongly inhibited methyl jasmonate-induced Vsp expression in soybean suspension-cultured cells. Both indole-3-acetic acid and NAA inhibited methyl jasmonate- and wound-induced expression of the Vsp and LoxA excised soybean trifoliate leaves and petioles. The less active auxin analog phenylacetic acid had less effect on methyl jasmonate- and wound-induced expression of these genes. Addition of cytokinin to alter the auxin:cytokinin ratio did not reverse auxin inhibition of Vsp expression. Transcription of [beta]-glucuronidase (Gus) modulated by a methyl jasmonate-responsive domain derived from the VspB promoter was minimally influenced by auxin. In contract, sucrose-induced expression of Gus mediated by a sucrose-responsive domain of the VspB promoter was strongly inhibited by NAA. We conclude that auxin inhibits Vsp mRNA accumulation, in part, by repressing sugar-mediated activation of Vsp expression.

Isolation and sequence analysis of proteins from mouse forebrain using two-dimensional gel electrophoresis coupled to high-pressure liquid extrusion.

This report presents a technique for recovery of mouse forebrain proteins from two-dimensional sodium dodecyl sulfate-polyacrylamide gels for subsequent primary structure determination. Proteins were visualized by Coomassie staining or salt precipitation and manually cut out of the gel. Excised spots were minced and loaded into an empty precolumn of a reversed-phase high-performance liquid chromatography system. Purified protein was extruded from a gel matrix by pressurized liquid, then separated from gel contaminants by reversed-phase gradient elution, and finally collected in siliconized tubes or on polybrene-coated filter disks for gas-phase sequencing. Several mouse and rat forebrain proteins were purified by this method and sequenced. Three previously unidentified mouse brain proteins with molecular weights of 4,000, 12,000, and 18,500 were partially sequenced and three hemoglobin fragments were structurally identified and mapped. Ribonuclease A, myoglobin, adrenocorticotropin, and bovine somatotropin were also subjected to two-dimensional (2-D) analysis and partially sequenced. Recovery values of 27-95% were obtained for extruded 14C-labeled ribonuclease, carbonic anhydrase, and bovine serum albumin out of sodium dodecyl sulfate-polyacrylamide gel electrophoretic gels. Losses resulting from the multiple handling steps of a 2-D gel separation process were also investigated. Recoveries of 12-17%, as determined by sequencing signals, were achieved. These latter recovery values reflect overall losses incurred in gel-focusing, gel-sizing, staining, destaining, high-pressure liquid extrusion, and N-terminal blockage. This work demonstrates that an array of protein spots can be systematically identified or defined by partial sequencing after high-pressure liquid extrusion from a 2-D gel matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

A nonurea electrophoretic gel system for resolution of polypeptides of Mr 2000 to Mr 200,000.

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system which resolves proteins and peptides from Mr 2000 to Mr 200,000 is described. Gradients of polyacrylamide, crosslinker, and glycerol buffered in Tris-phosphate (pH 6.8) are employed. Neither urea nor a stacking gel is required. This system has been used to separate molecules below Mr 3000 which differed by only seven amino acid residues, yet has the capacity to survey masses up to Mr 200,000 on the same gel. Examples are given for separations of myoglobin cyanogen bromide fragments and adrenocorticotropin peptides. Utilizing the same gradient slab gel system in tandem with isoelectric focusing, a two-dimensional separation pattern of mammalian liver cell lysate is shown. A comparison of two different silver stain methods with this system is also given.

The soybean vegetative storage proteins VSP alpha and VSP beta are acid phosphatases active on polyphosphates.

The soybean vegetative storage protein genes (vspA, and vspB) are regulated in a complex manner developmentally and in response to external stimuli such as wounding and water deficit. The proteins accumulate to almost one-half the amount of soluble leaf protein when soybean plants are continually depodded and have been identified as storage proteins because of their abundance and pattern of expression in plant tissues. We have shown that purified VSP homodimers (VSP alpha and VSP beta) and heterodimers (VSP alpha/beta) possess acid phosphatase activity (alpha = 0.3-0.4 units/mg; beta = 2-4 units/mg; alpha/beta = 7-10 units/mg). Specific activities were determined by monitoring o-carboxyphenyl phosphate (0.7 mM) cleavage at pH 5.5 (VSP alpha) or pH 5.0 (VSP alpha/beta and VSP beta) in 0.15 M sodium acetate buffer at 25 degrees C. These enzymes are active over a broad pH range, maintaining greater than 40% of maximal activity from pH 4.0 to 6.5 and having maximal activity at pH 5.0-5.5. They are inactivated by sodium fluoride, sodium molybdate, and heating at 70 degrees C for 10 min. These phosphatases can liberate Pi from several different substrates, including napthyl acid phosphate, carboxyphenyl phosphate, sugar-phosphates, glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, ATP, ADP, PPi, and short chain polyphosphates. VSP alpha/beta cleaved phosphoenolpyruvate, ATP, ADP, PPi, and polyphosphates most efficiently. Apparent Km and Vmax values at 25 degrees C and pH 5.0 were 42 microM and 2.0 mumol/min/mg, 150 microM and 4.2 mumol/min/mg, and 420 microM and 4.1 mumol/min/mg, for tetrapolyphosphate, pyrophosphate, and phosphoenolpyruvate, respectively.

Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology.

We have cloned the gene, vps34+, from the fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. pombe Vps34 protein shares 43% amino acid sequence identity with the Saccharomyces cerevisiae Vps34 protein and 28% identity with the p110 catalytic subunit of the mammalian phosphatidylinositol 3-kinase. When the vps34+ gene is disrupted, S.pombe strains are temperature-sensitive for growth and the mutant cells contain enlarged vacuoles. Furthermore, while wild-type strains exhibit substantial levels of phosphatidylinositol 3-kinase activity, this activity is not detected in the vps34 delta strain. S.pombe Vps34p-specific antiserum detects a single protein in cells of -90 kDa that fractionates almost exclusively with the crude membrane fraction. Phosphatidylinositol 3-kinase activity also is localized mainly in the membrane fraction of wild-type cells. Immunoisolated Vps34p specifically phosphorylates phosphatidylinositol on the D-3 position of the inositol ring to yield phosphatidylinositol(3)phosphate. but does not utilize phosphatidylinositol(4)phosphate or phosphatidylinositol(4,5)bisphosphate as substrates. In addition, when compared to the mammalian p110 phosphatidylinositol 3-kinase, S. pombe Vps34p is relatively insensitive to the inhibitors wortmannin and LY294002. Together, these results indicate that S. pombe Vps34 is more similar to the phosphatidylinositol-specific 3-kinase, Vps34p from S. cerevisiae, and is distinct from the p110/p85 and G protein-coupled phosphatidylinositol 3-kinases from mammalian cells. These data are discussed in relation to the possible role of Vps34p in vesicle-mediated protein sorting to the S. pombe vacuole.

Analysis of phosphoinositides in protein trafficking.

Phosphoinositides are key regulators of vesicle-mediated protein trafficking. Their roles include recruiting vesicle coat and effector proteins to the site of budding and promoting vesicle fusion. The intracellular levels of phosphoinositides and their localization to intracellular membranes are critical to their functions. An analytical procedure was developed that optimizes the recovery of radiolabeled cellular phosphoinositides. Quantitative analyses of yeast cellular phosphoinositides indicated that this approach is useful for examining the intracellular membrane phosphoinositide compositions related to trafficking phenomena. The approach will also enable investigators to determine whole-plant phosphoinositide compositions that have been difficult to achieve in the past. These analytical advances should be generally applicable to studies of phosphoinositide dynamics related to membrane trafficking in yeast, plant, and animal cells.

Ceramide dissociates 3'-phosphoinositide production from pleckstrin homology domain translocation.

Numerous hormones, cytokines and transforming oncogenes activate phosphoinositide 3-kinase (PI-3K), a lipid kinase that initiates signal transduction cascades regulating cellular proliferation, survival, protein synthesis and glucose metabolism. PI-3K catalyses the production of the 3'-phosphoinositides PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3), which recruit downstream effector enzymes to the membrane via their pleckstrin homology (PH) domains. Recent studies have indicated that another signalling lipid, the sphingolipid ceramide, inhibits several PI-3K-dependent events, including insulin-stimulated glucose uptake and growth-factor-stimulated cell survival. Here we show that ceramide analogues specifically prevent the recruitment of the PtdIns(3,4,5)P(3)-binding proteins Akt/protein kinase B (PKB) or the general receptor for phosphoinositides-1 (GRP1). Specifically, the short-chain ceramide derivative C2-ceramide inhibited the platelet-derived growth factor (PDGF)-stimulated translocation of full-length Akt/PKB, as well as truncated proteins encoding only the PH domains of Akt/PKB or GRP1. C2-ceramide did not alter the membrane localization of the PH domain for phospholipase Cdelta, which preferentially binds PtdIns(4,5)P(2), nor did it affect the PDGF-stimulated production of PtdIns(3,4)P(2) or PtdIns(3,4,5)P(3). Interestingly, a glucosylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol (PDMP), shown previously to increase intracellular ceramide concentrations without affecting PI-3K [Rani, Abe, Chang, Rosenzweig, Saltiel, Radin and Shayman (1995) J. Biol. Chem. 270, 2859-2867], recapitulated the inhibitory effects of C2-ceramide on PDGF-stimulated Akt/PKB phosphorylation. These studies indicate that ceramide prevents the translocation of certain PtdIns(3,4,5)P(3)-binding proteins, despite the presence of a full complement of PtdIns(3,4)P(2) or PtdIns(3,4,5)P(3). Furthermore, these findings suggest a mechanism by which stimuli that induce ceramide synthesis could negate the fundamental signalling pathways initiated by PI-3K.

Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter.

Soybean vspB encodes a highly expressed vegetative storage protein-acid phosphatase. In soybean, vspB expression is stimulated by methyl jasmonate (MeJA) and sugars. The vspB promoter was studied by transforming tobacco with fusions of 5' noncoding vspB DNA and the gene encoding beta-glucuronidase (GUS). Constructs containing 833 bp of vspB 5' DNA showed high expression of GUS in stems, leaf veins and trichomes, sepals, and pollen. Sucrose (0.2 M) and MeJA (10(-5) M) increased gene expression when applied to leaf tissue. Deletion of the region -787 to -520 with respect to the transcription initiation site rendered the vspB promoter noninducible by MeJA but still sucrose responsive. This result indicates that DNA elements capable of modulating vspB by MeJA can be separated from carbon response elements. Further 5' end deletion from -520 to -403 or 3' end deletion from -165 to -289 removed DNA sequences involved in carbon modulation of gene expression. A DNA domain that mediates the MeJA response was further localized to a 50-bp region between -535 and -585. This domain when fused to a cauliflower mosaic virus (CaMV) 35S truncated (-88) promoter makes the CaMV promoter responsive to MeJA. The MeJA-responsive domain contains a G-box motif (CACGTG) and a C-rich sequence. A similar 50-bp DNA region is present in the putative promoter of vspA. Related sequences are located in a wound- and MeJA-responsive domain of the proteinase inhibitor II gene and a UV-responsive promoter domain of chs, the gene encoding chalcone synthase that is also responsive to MeJA.

Separation of protein hormones.

The purpose of this review is to highlight modern techniques in HPLC and electrophoresis used for protein hormone separations. The advent of biotechnological methods for production of synthetic polypeptides and recombinant proteins will have a significant future impact on the types of therapeutics and metabolites that need to be monitored in the clinical laboratory. The protein hormone examples given in this work were selected because of the comprehensive body of separation science literature and not necessarily for their future importance in medicine. The intention was to present an array of general methods and techniques which may be useful to the clinical investigator for analysis of any protein hormone.

Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars.

The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves.

Intracellular delivery of phosphoinositides and inositol phosphates using polyamine carriers.

Phosphoinositide signaling regulates events in endocytosis and exocytosis, vesicular trafficking of proteins, transduction of extracellular signals, remodeling of the actin cytoskeleton, regulation of calcium flux, and apoptosis. Obtaining mechanistic insights in living cells is impeded by the membrane impermeability of these anionic lipids. We describe a carrier system for intracellular delivery of phosphoinositide polyphosphates (PIP(n)s) and fluorescently labeled PIP(n)s into living cells, such that intracellular localization can be directly observed. Preincubation of PIP(n)s or inositol phosphates with carrier polyamines produced complexes that entered mammalian, plant, yeast, bacterial, and protozoal cells in seconds to minutes via a nonendocytic mechanism. Time-dependent transit of both PIP(n)s and the carrier to specific cytosolic and nuclear compartments was readily visualized by fluorescence microscopy. Platelet-derived growth factor treatment of NIH 3T3 fibroblasts containing carrier-delivered phosphatidylinositol 4,5-bisphosphate [PtdIns(4, 5)P(2)]-7-nitrobenz-2-oxa-1,3-diazole resulted in the redistribution of the fluorescent signal, suggesting that fluorescent PtdIns(4, 5)P(2) was a substrate for phospholipase C. We also observed a calcium flux in NIH 3T3 cells when complexes of carrier and PtdIns(4, 5)P(2) or inositol 1,4,5-trisphosphate were added extracellularly. This simple intracellular delivery system allows for the efficient translocation of biologically active PIP(n)s, inositol phosphates, and their fluorescent derivatives into living cells in a physiologically relevant context.

Hyperosmotic stress induces the rapid phosphorylation of a soybean phosphatidylinositol transfer protein homolog through activation of the protein kinases SPK1 and SPK2.

Although phosphatidylinositol transfer proteins (PITPs) are known to serve critical functions in regulating a varied array of signal transduction processes in animals and yeast, the discovery of a similar class of proteins in plants occurred only recently. Here, we report the participation of Ssh1p, a soybean PITP-like protein, in the early events of osmosensory signal transduction in plants, a function not attributed previously to animal or yeast PITPs. Exposure of plant tissues to hyperosmotic stress led to the rapid phosphorylation of Ssh1p, a modification that decreased its ability to associate with membranes. An osmotic stress-activated Ssh1p kinase activity was detected in several plant species by presenting recombinant Ssh1p as a substrate in in-gel kinase assays. Elements of a similar osmosensory signaling pathway also were conserved in yeast, an observation that facilitated the identification of soybean protein kinases SPK1 and SPK2 as stress-activated Ssh1p kinases. This study reveals the activation of SPK1 and/or SPK2 and the subsequent phosphorylation of Ssh1p as two early successive events in a hyperosmotic stress-induced signaling cascade in plants. Furthermore, Ssh1p is shown to enhance the activities of a plant phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase, an observation that suggests that the ultimate function of Ssh1p in cellular signaling is to alter the plant's capacity to synthesize phosphoinositides during periods of hyperosmotic stress.

A role for host phosphoinositide 3-kinase and cytoskeletal remodeling during Cryptosporidium parvum infection.

Cryptosporidium parvum preferentially infects epithelial cells lining the intestinal mucosa of mammalian hosts. Parasite development and propagation occurs within a unique intracellular but extracytoplasmic parasitophorous vacuole at the apical surface of infected cells. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured bovine fallopian tube epithelial (BFTE) cells inoculated with C. parvum sporozoites. Indirect-immunofluorescence microscopy detected host tyrosine phosphorylation within 30 s of inoculation. At >30 min postinoculation, actin aggregates were detected at the site of parasite attachment by fluorescein isothiocyanate-conjugated phalloidin staining as well as by indirect immunolabeling with monoclonal anti-actin. The actin-binding protein villin was also detected in focal aggregates at the site of attachment. Host cytoskeletal rearrangement persisted for the duration of the parasitophorous vacuole and contributed to the formation of long, branched microvilli clustered around the cryptosporidial vacuole. The phosphoinositide 3-kinase inhibitor wortmannin significantly inhibited (P < 0.05) C. parvum infection when BFTE cells were pretreated for 60 min at 37 degreesC prior to inoculation. Similarly, treatment of BFTE cells with the protein kinase inhibitors genistein and staurosporine and the cytoskeletally acting compounds 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine, cytochalasin D, and 2,3-butanedione monoxime significantly inhibited (P < 0.05) in vitro infection at 24 h postinoculation. These findings demonstrate a prominent role for phosphoinositide 3-kinase activity during the early C. parvum infection process and suggest that manipulation of host signaling pathways results in actin rearrangement at the site of sporozoite attachment.