Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.     

Differential regulation by phosphatidylinositol 4,5-bisphosphate of pituitary plasma-membrane and cytosolic phosphoinositide kinases.

Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.     

Regulation of plasma membrane Ca2+ ATPases by lipids of the phosphatidylinositol cycle

When the erythrocyte plasma membrane Ca2+ pump is reconstituted into phosphatidylcholine liposomes, the inclusion of small amounts of phosphatidic acid or phosphatidylinositol 4,5-bisphosphate stimulates the enzyme's activity. Other lipids of the phosphatidylinositol cycle (diacylglycerol, phosphatidylinositol) have little effect. The stimulatory effect of phosphatidylinositol 4,5-bisphosphate is greater than that of calmodulin; this lipid also stimulates the plasma membrane Ca2+ ATPase from rat brain.     

Phospholipase D

Phospholipase D catalyses the hydrolysis of phosphatidylcholine to generate phosphatidate. The regulation of PLD activity is complex involving a number of small GTP binding proteins, but in particular Arf and Rho, phosphatidylinositol 4,5-bisphosphate and protein kinase C. The cDNA for PLD1 has recently been cloned and shows homology to the yeast and plant genes but only within four domains. Domains I and IV each contain a putative catalytic triad. PLD activity has been detected in plasma membranes, Golgi membranes and in nuclear membranes; it is unclear if different isoenzymes are responsible for this variation, or if the PLDs are differently regulated. The product of PLD activity, PA, appears to be a messenger molecule regulating the actin cytoskeleton and maybe playing a role in the control of membrane traffic and secretion.     

Rho small GTPases activate the epithelial Na(+) channel

Small G proteins in the Rho family are known to regulate diverse cellular processes, including cytoskeletal organization and cell cycling, and more recently, ion channel activity and activity of phosphatidylinositol 4-phosphate 5-kinase (PI(4)P 5-K). The present study investigates regulation of the epithelial Na(+) channel (ENaC) by Rho GTPases. We demonstrate here that RhoA and Rac1 markedly increase ENaC activity. Activation by RhoA was suppressed by the C3 exoenzyme. Inhibition of the downstream RhoA effector Rho kinase, which is necessary for RhoA activation of PI(4)P 5-K, abolished ENaC activation. Similar to RhoA, overexpression of PI(4)P 5-K increased ENaC activity suggesting that production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in response to RhoA-Rho kinase signaling stimulates ENaC. Supporting this idea, inhibition of phosphatidylinositol 4-kinase, but not the RhoA effector phosphatidylinositol 3-kinase and MAPK cascades, markedly attenuated RhoA-dependent activation of ENaC. RhoA increased ENaC activity by increasing the plasma membrane levels of this channel. We conclude that RhoA activates ENaC via Rho kinase and subsequently activates PI(4)P 5-K with concomitant increases in PI(4,5)P(2) levels promoting channel insertion into the plasma membrane.     

Subcellular localisation of inositol lipid kinases in rat liver

The subcellular distribution of the enzymes which phosphorylate phosphatidylinositol sequentially to form phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was investigated in rat liver. We demonstrate that whilst phosphatidylinositol kinase is present in Golgi, lysosomes and plasma membranes, the kinase that forms phosphatidylinositol 4,5-bisphosphate is localised predominantly at the plasma membrane. The role of the inositol lipid kinases in cell function is discussed.     

Phosphatidylinositol 4,5-bisphosphate formation in rabbit skeletal and heart muscle membranes

Incubation of rabbit skeletal muscle microsomes or isolated triads with gamma 32P-ATP/Mg2+ in the absence and in the presence of added phosphatidylinositol resulted in the formation of phosphatidylinositol 4-phosphate catalyzed by phosphatidylinositol kinase. When phosphatidylinositol 4-phosphate was added as exogenous substrate, phosphatidylinositol 4,5-bisphosphate was also formed demonstrating the presence of a membrane bound phosphatidylinositol 4-phosphate kinase. Triads were broken mechanically in a French press and separated on a continuous sucrose gradient. Incubation of these fractions with gamma 32P-ATP/Mg2+ resulted in a rapid labeling of phospholipid in a membrane fraction banding between transverse tubules and the terminal cisternae. Partial triad breakage and triad reformation experiments indicated that this phosphatidylinositol kinase was associated with T-tubules. When exogenous phosphatidylinositol 4-phosphate was employed as substrate phosphatidylinositol 4,5-bisphosphate and phosphatidic acid were formed, indicating the presence of all the enzymes of the polyphosphoinositide signaling system in this special membrane fraction. In contrast, heart muscle microsomes or plasma membranes can catalyze this reaction sequence from endogenous formed phosphatidylinositol 4-phosphate.     

Selective activation of phospholipase D2 by unsaturated fatty acid.

Although oleate has been implicated in the regulation of phospholipase D (PLD) activity, the molecular identity of the oleate-stimulated PLD is still poorly understood. We now report that oleate selectively stimulates the enzymatic activity of PLD2 but not of PLD1, with an optimal concentration of 20 microM in vitro. Intriguingly, phosphatidylinositol 4,5-bisphosphate (PIP2) synergistically stimulates the oleate-dependent PLD2 activity with an optimal concentration of 2.5 microM. These results provide the first evidence that oleate is a PLD2-specific activating factor and PLD2 activity is synergistically stimulated by oleate and PIP2.     

Interaction of the type Ialpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4, 5-bisphosphate in the regulation of PLD2 activity

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane trafficking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P(2): phosphatidylinositol 4-phosphate 5-kinase alpha (Type Ialpha PIPkinase). We find that both PLD1 and PLD2 interact with the Type Ialpha PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Ialpha PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P(2) is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid.     

A role for rho-kinase in rho-controlled phospholipase D stimulation by the m3 muscarinic acetylcholine receptor.

Stimulation of phospholipase D (PLD) by membrane receptors is now recognized as a major signal transduction pathway involved in diverse cellular functions. Rho proteins control receptor signaling to PLD, and these GTPases have been shown to directly stimulate purified recombinant PLD1 enzymes in vitro. Here we report that stimulation of PLD activity, measured in the presence of phosphatidylinositol 4,5-bisphosphate, by RhoA in membranes of HEK-293 cells expressing the m3 muscarinic acetylcholine receptor (mAChR) is phosphorylation-dependent. Therefore, the possible involvement of the RhoA-stimulated serine/threonine kinase, Rho-kinase, was investigated. Overexpression of Rho-kinase and constitutively active Rho-kinase (Rho-kinase-CAT) but not of kinase-deficient Rho-kinase-CAT markedly increased m3 mAChR-mediated but not protein kinase C-mediated PLD stimulation, similar to overexpression of RhoA. Expression of the Rho-inactivating C3 transferase abrogated the stimulatory effect of wild-type Rho-kinase, but not of Rho-kinase-CAT. Recombinant Rho-kinase-CAT mimicked the phosphorylation-dependent PLD stimulation by RhoA in HEK-293 cell membranes. Finally, the Rho-kinase inhibitor HA-1077 largely inhibited RhoA-induced PLD stimulation in membranes as well as PLD stimulation by the m3 mAChR but not by protein kinase C in intact HEK-293 cells. We conclude that Rho-kinase is involved in Rho-dependent PLD stimulation by the G protein-coupled m3 mAChR in HEK-293 cells. Thus, our findings identify Rho-kinase as a novel player in the receptor-controlled PLD signaling pathway.     

Regulation of phospholipase D by phosphorylation-dependent mechanisms.

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4, 5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Calpha as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.     

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.     

Identification of centaurin-alpha2: a phosphatidylinositide-binding protein present in fat,heart and skeletal muscle

We describe here the cloning, expression and characterisation of centaurin-alpha2 from a rat adipocyte cDNA library. The centaurin-alpha2 cDNA contains an open reading frame, which codes for a protein of 376 amino acids with predicted mass of 43.5 kDa. Centaurin-alpha2 shares 51-59% identity with centaurin-alpha1 proteins and has the same domain organisation, consisting of a predicted N-terminal ArfGAP domain followed by two successive pleckstrin homology domains. Despite the sequence similarity, there are a number of notable differences between the previously characterised centaurin-alpha1 proteins and the newly described centaurin-alpha2: (i) in vitro lipid binding experiments with centaurin-alpha2 do not reveal the same selectivity for phosphatidylinositol 3,4,5-trisphosphate over phosphatidylinositol 4,5-bisphosphate that has been shown for centaurin-alpha; (ii) unlike centaurin-alpha1 which is expressed mainly in the brain, centaurin-alpha2 has a broad tissue distribution, being particularly abundant in fat, heart and skeletal muscle; (iii) in contrast to centaurin-alpha1 which is found in both membrane and cytosolic fractions, endogenous centaurin-alpha2 is exclusively present in the dense membrane fractions of cell extracts, suggesting a constitutive membrane association. Insulin stimulation, which stimulates phosphatidylinositol 3,4,5-trisphosphate production, does not alter the subcellular distribution of centaurin-alpha2 between adipocyte membrane fractions. This observation is consistent with the lack of specificity of centaurin-alpha2 for phosphatidylinositol 3,4,5-trisphosphate over phosphatidylinositol 4,5-bisphosphate.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

Regulation of phospholipase D by phosphorylation-dependent mechanisms

The rapid production of phosphatidic acid following receptor stimulation has been demonstrated in a wide range of mammalian cells. Virtually every cell uses phosphatidylcholine as substrate to produce phosphatidic acid in a controlled reaction catalyzed by specific PLD isoforms. Considerable effort has been directed at studying the regulation of PLD activities and subsequent work has characterized a family of proteins including PLD1 and PLD2. Whereas both PLD enzymes are dependent on phosphatidylinositol 4,5-bisphosphate for activity only the PLD1 isoform was strongly stimulated by the small GTPases ARF and RhoA and by protein kinase Cα as well. A role for tyrosine kinase activities in the membrane recruitment of small GTPases, in the synthesis of phosphatidylinositol 4,5-bisphosphate and tyrosine phosphorylation of PLD1 and PLD2 has been uncovered. However, it still not clear exactly how tyrosine phosphorylation of proteins contributes to PLD activation in cells. Here we review the data linking tyrosine phosphorylation of proteins to the activation of PLD and describe recent finding on the sites and possible mechanisms of action of tyrosine kinases in receptor-mediated PLD activation. Finally, a model illustrating the potential complex interplay linking these signaling events with the activation of PLD is presented.     

Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKCalpha in RBL-2H3 cells

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCalpha at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase-dependent stimulation of Rac1, ARF6, and PKCalpha.     

Differential phospholipase D activation by bradykinin and sphingosine 1-phosphate in NIH 3T3 fibroblasts overexpressing gelsolin.

Gelsolin, an actin-binding protein, shows a strong ability to bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here we showed in in vitro experiments that gelsolin inhibited recombinant phospholipase D1 (PLD1) and PLD2 activities but not the oleate-dependent PLD and that this inhibition was not reversed by increasing PIP(2) concentration. To investigate the role of gelsolin in agonist-mediated PLD activation, we used NIH 3T3 fibroblasts stably transfected with the cDNA for human cytosolic gelsolin. Gelsolin overexpression suppressed bradykinin-induced activation of phospholipase C (PLC) and PLD. On the other hand, sphingosine 1-phosphate (S1P)-induced PLD activation could not be modified by gelsolin overexpression, whereas PLC activation was suppressed. PLD activation by phorbol myristate acetate or Ca(2+) ionophore A23187 was not affected by gelsolin overexpression. Stimulation of control cells with either bradykinin or S1P caused translocation of protein kinase C (PKC) to the membranes. Translocation of PKC-alpha and PKC-beta1 but not PKC-epsilon was reduced in gelsolin-overexpressed cells, whereas phosphorylation of mitogen-activated protein kinase was not changed. S1P-induced PLC activation and mitogen-activated protein kinase phosphorylation were sensitive to pertussis toxin, but PLD response was insensitive to such treatment, suggesting that S1P induced PLD activation via certain G protein distinct from G(i) for PLC and mitogen-activated protein kinase pathway. Our results suggest that gelsolin modulates bradykinin-mediated PLD activation via suppression of PLC and PKC activities but did not affect S1P-mediated PLD activation.     

Roles of phosphoinositides and of Spo14p (phospholipase D)-generated phosphatidic acid during yeast sporulation

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P2 synthesis.     

Generation of phosphatidylinositol 4,5-bisphosphate proceeds through an intracellular route in rat hepatocytes

The subcellular distribution in rat hepatocytes of enzymes participating in the entire generation cycle of phosphatidylinositol 4,5-bisphosphate, and phosphorylated intermediates of this pathway, has been examined by Nycodenz gradient centrifugation. Our results indicate that the synthesis of phosphatidylinositol takes place in the endoplasmic reticulum, and that its phosphorylation to phosphatidylinositol 4-phosphate occurs intracellularly in low-density membranes before translocation to the plasma membrane, where it is further phosphorylated to phosphatidylinositol 4,5-bisphosphate. The intracellular formation of PIP implies a vesicular transport to the plasma membrane.