SPO14 separation-of-function mutations define unique roles for phospholipase D in secretion and cellular differentiation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, phospholipase D (PLD), encoded by the SPO14 gene, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phosphatidic acid. SPO14 is essential for cellular differentiation during meiosis and is required for Golgi function when the normal secretory apparatus is perturbed (Sec14-independent secretion). We isolated specific alleles of SPO14 that support Sec14-independent secretion but not sporulation. Identification of these separation-of-function alleles indicates that the role of PLD in these two physiological processes is distinct. Analyses of the mutants reveal that the corresponding proteins are stable, phosphorylated, catalytically active in vitro, and can localize properly within the cell during meiosis. Surprisingly, the separation-of-function mutations map to the conserved catalytic region of the PLD protein. Choline and phosphatidic acid molecular species profiles during Sec14-independent secretion and meiosis reveal that while strains harboring one of these alleles, spo14S-11, hydrolyze phosphatidylcholine in Sec14-independent secretion, they fail to do so during sporulation or normal vegetative growth. These results demonstrate that Spo14 PLD catalytic activity and cellular function can be differentially regulated at the level of phosphatidylcholine hydrolysis.

Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes.

Phosphoinositides are both substrates for second messenger-generating enzymes and spatially localized membrane signals that mediate vital steps in signal transduction, cytoskeletal regulation and membrane trafficking. Phosphatidylcholine-specific phospholipase D (PLD) activity is stimulated by phosphoinositides, but the mechanism and physiological requirement for such stimulation to promote PLD-dependent cellular processes is not known. To address these issues, we have identified a site at which phosphoinositides interact with PLD and have assessed the role of this region in PLD function. This interacting motif contains critical basic amino acid residues that are required for stimulation of PLD activity by phosphoinositides. Although PLD alleles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane-associated and properly localized within the cell but are inactive against cellular lipid substrates. Analogous mutations of this site in yeast PLD, Spo14p, result in enzymes that localize normally, but with catalytic activity that has dramatically reduced responsiveness to phosphoinositides. The level of responsiveness to phosphoinositides in vitro correlated with the ability of PLD to function in vivo. Taken together, these results provide the first evidence that phosphoinositide regulation of PLD activity observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.

Regulation and function of PLDs in yeast.

While yeast contain multiple phospholipase D activities, only one, encoded by SPO14, appears to be a member of the phosphatidylcholine-specific phospholipase D gene family. Genetic analyses have revealed a role for this enzyme in regulated membrane trafficking events.

ADP-Ribosylation factors do not activate yeast phospholipase Ds but are required for sporulation.

ADP-ribosylation factor (ARF) proteins in Saccharomyces cerevisiae are encoded by two genes, ARF1 and ARF2. The addition of the c-myc epitope at the C terminus of Arf1 resulted in a mutant (arf1-myc arf2) that supported vegetative growth and rescued cells from supersensitivity to fluoride, but homozygous diploids failed to sporulate. arf1-myc arf2 mutants completed both meiotic divisions but were unable to form spores. The SPO14 gene encodes a phospholipase D (PLD), whose activity is essential for mediating the formation of the prospore membrane, a prerequisite event for spore formation. Spo14 localized normally to the developing prospore membrane in arf1-myc arf2 mutants; however, the synthesis of the membrane was attenuated. This was not a consequence of reduced PLD catalytic activity, because the enzymatic activity of Spo14 was unaffected in meiotic arf1-myc arf2 mutants. Although potent activators of mammalian PLD1, Arf1 proteins did not influence the catalytic activities of either Spo14 or ScPld2, a second yeast PLD. These results demonstrate that ARF1 is required for sporulation, and the mitotic and meiotic functions of Arf proteins are not mediated by the activation of any known yeast PLD activities. The implications of these results are discussed with respect to current models of Arf signaling.

Relocalization of phospholipase D activity mediates membrane formation during meiosis.

Phospholipase D (PLD) enzymes catalyze the hydrolysis of phosphatidylcholine and are involved in membrane trafficking and cytoskeletal reorganization. The Saccharomyces cerevisiae SPO14 gene encodes a PLD that is essential for meiosis. We have analyzed the role of PLD in meiosis by examining two mutant proteins, one with a point mutation in a conserved residue (Spo14pK--> H) and one with an amino-terminal deletion (Spo14pDeltaN), neither of which can restore meiosis in a spo14 deletion strain. Spo14pK--> H is enzymatically inactive, indicating that PLD activity is required, whereas Spo14pDeltaN retains PLD catalytic activity in vitro, indicating that PLD activity is not sufficient for meiosis. To explore other aspects of Spo14 function, we followed the localization of the enzyme during meiosis. Spo14p is initially distributed throughout the cell, becomes concentrated at the spindle pole bodies after the meiosis I division, and at meiosis II localizes to the new spore membrane as it surrounds the nuclei and then expands to encapsulate the associated cytoplasm during the formation of spores. The catalytically inactive protein also undergoes relocalization during meiosis; however, in the absence of PLD activity, no membrane is formed. In contrast, Spo14pDeltaN does not relocalize properly, indicating that the failure of this protein to complement a spo14 mutant is due to its inability to localize its PLD activity. Furthermore, we find that Spo14p movement is correlated with phosphorylation of the protein. These experiments indicate that PLD participates in regulated membrane formation during meiosis, and that both its catalytic activity and subcellular redistribution are essential for this function.

Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity.

Phospholipase D (PLD) genes are members of a superfamily that is defined by several highly conserved motifs. PLD in mammals has been proposed to play a role in membrane vesicular trafficking and signal transduction. Using site-directed mutagenesis, 25 point mutants have been made in human PLD1 (hPLD1) and characterized. We find that a motif (HxKxxxxD) and a serine/threonine conserved in all members of the PLD superfamily are critical for PLD biochemical activity, suggesting a possible catalytic mechanism. Functional analysis of catalytically inactive point mutants for yeast PLD demonstrates that the meiotic phenotype ensuing from PLD deficiency in yeast derives from a loss of enzymatic activity. Finally, mutation of an HxKxxxxD motif found in a vaccinia viral protein expressed in the Golgi complex results in loss of efficient vaccinia virus cell-to-cell spreading, implicating the viral protein as a member of the superfamily and suggesting that it encodes a lipid modifying or binding activity. The results suggest that vaccinia virus and hPLD1 may act through analogous mechanisms to effect viral cellular egress and vesicular trafficking, respectively.

Human ADP-ribosylation factor-activated phosphatidylcholine-specific phospholipase D defines a new and highly conserved gene family.

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been implicated as a critical step in numerous cellular pathways, including signal transduction, membrane trafficking, and the regulation of mitosis. We report here the identification of the first human PLD cDNA, which defines a new and highly conserved gene family. Characterization of recombinant human PLD1 reveals that it is membrane-associated, selective for phosphatidylcholine, stimulated by phosphatidylinositol 4,5-bisphosphate, activated by the monomeric G-protein ADP-ribosylation factor-1, and inhibited by oleate. PLD1 likely encodes the gene product responsible for the most widely studied endogenous PLD activity.

The pleckstrin homology domain of phospholipase C-delta 1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes.

The pleckstrin homology (PH) domain of phospholipase C-delta 1 (PLC-delta 1) binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in phospholipid membranes with an affinity (Ka approximately 10(6) M-1) and specificity comparable to those of the native enzyme. PLC-delta 1 and its PH domain also bind inositol 1,4,5-trisphosphate, the polar head group of PI(4,5)P2, with comparable affinity and approximately 1:1 stoichiometry. A peptide corresponding to amino acids 30-43 of the PLC-delta 1 PH domain contains several basic residues predicted to bind PI(4,5)P2, but binds weakly and with little specificity for PI(4,5)P2; hence the tertiary structure of the isolated PH domain is required for high affinity PI(4,5)P2 binding. Our PI-(4,5)P2 binding results support the hypothesis that the intact PH domain, serving as a specific tether, directs PLC-delta 1 to membranes enriched in PI(4,5)P2 and permits the active site, located elsewhere in the protein, to hydrolyze multiple substrate molecules before this enzyme dissociates from the membrane surface.

Phospholipase D signaling is essential for meiosis.

Phospholipid metabolism plays an important role in cellular regulation by generating second messengers for signal transduction. Many stimuli activate a phospholipase D, which catalyzes the hydrolysis of phosphatidylcholine, producing phosphatidic acid and choline. Here we report that the yeast SP014 gene, which is essential for meiosis [Honigberg, S. M., Conicella, C. & Esposito, R. E. (1992) Genetics 130, 703-716], encodes a phospholipase D. SP014 RNA and protein activity are induced during late meiotic prophase, and the enzyme has properties similar to mammalian phosphatidylinositol 4,5-bisphosphate-regulated phospholipase D. Characterization of an unusual allele of SP014 defines regions of the protein important for enzyme catalysis and regulation. These results implicate phospholipase D signaling in regulating cellular differentiation.

Regulation of phosphoinositide-3-kinase by G protein beta gamma subunits in a rat osteosarcoma cell line.

Rat osteosarcoma 17/2.8 cells (Ros 17/2.8 cells) were labeled with [32P]PO4(2-), and their levels of inositol lipids were determined after stimulation with thrombin. Thrombin stimulated a pertussis toxin-sensitive rapid accumulation of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3] with lesser increases in levels of phosphatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol-3-phosphate [PtdIns3P] that were slower in onset. Ros 17/2.8 cell homogenates contained phosphatase activities that hydrolyzed PtdIns(3,4,5)P3 to PtdIns(3,4)P2 and PtdIns3P. Phosphoinositide-3-kinase activity was determined in Ros 17/2.8 cell homogenates using exogenously provided PtdIns(4,5)P2. Guanosine-5'-3-O-(thio)triphosphate caused an approximately 3-fold increase in phosphoinositide-3-kinase activity in a manner that was blocked by high concentrations of guanosine-5'-2-O-(thio)diphosphate. Purified bovine brain G protein beta gamma subunits also increased phosphoinositide-3-kinase activity modestly in Ros 17/2.8 cell homogenates. Ros 17/2.8 cell homogenates contained phosphatase activities that sequentially dephosphorylated PtdIns(3,4,5)P3 to PtdIns(3,4)P2 and PtdIns3P. Two peaks of phosphoinositide-3-kinase activity were resolved by anion exchange chromatography of a Ros 17/2.8 cell cytosolic extract. The later elution of these was selectively activated by beta gamma subunits (16-fold activation with 16 microM beta gamma subunits). Half-maximal effects of the beta gamma subunits were observed at a concentration of 0.6 microM, and activation was blocked by preincubation of the beta gamma subunits with an excess of recombinant Gi alpha 2. beta gamma Subunits did not activate the p85 alpha/p110 beta form of phosphoinositide-3-kinase purified from sf9 cells after expression with the use of baculovirus vectors.

Inositol lipid-mediated signalling in response to endothelin and ATP in the mammalian testis.

The testis is a complex organ in which local control is achieved by signalling between its constituent cells. Herein we describe the responses of cultured rat testicular cells and a mouse Sertoli cell-line to stimulation by endothelin and ATP, and elsewhere we have shown that rat peritubular myoid cells possess phosphoinositidase C-coupled V1a-vasopressin receptors identical to those of liver (Howl, J. et al, 1995, Endocrinology 136: 2206-2213). 1. Peritubular myoid cells from pre-pubertal rats responded through ETA receptors with PtdIns(4,5)P2 hydrolysis [EC50 for endothelin-1 (ET-1) approximately 0.4 nM], elevation of intracellular [Ca2+], and tyrosine phosphorylation of a variety of cellular proteins. They also showed enhanced adenylate cyclase activity, with an EC50 for ET-1 of approximately 3 nM, also through ETA receptors. Pharmacological elevation of [cAMP] did not immediately change the ET-1-stimulated formation of inositol phosphates, but attenuated the response after several hours. 2. Pre-pubertal rat Sertoli cells showed no detectable responses to ET-1, but responded to FSH with elevated [cAMP] and to ATP with PtdIns(4,5)P2 hydrolysis. PtdIns(4,5)P2 hydrolysis was equally responsive to ATP and UTP, and so appears to be activated by P2U-purinergic receptors. This response was enhanced by protein kinase C inhibition and attenuated by PKC activation. 3. Despite its lack of effect on rat Sertoli cells in primary culture, ET-1 provoked PtdIns(4,5)P2 hydrolysis in the TM4 murine Sertoli cell line (EC50 approximately 0.6 nM), and this response was negatively regulated by protein kinase C activation. 5. No receptor-stimulated activation of phosphoinositase C was detected in 'germ cell' populations, but the non-specific G protein activator A1F4-provoked inositol phosphate accumulation in these cells, so demonstrating their potential to respond through yet to be identified G protein-coupled receptors with phosphoinositidase C activation. 6. Immunoblotting studies showed the presence in rat testis of phosphoinositidase C-beta 1 and the alpha-subunits(s) of the G-protein(s) Gq and/or G11. These studies show that testicular myoid and Sertoli cells use at least three G protein-coupled receptors (V1a-vasopressins, ETA-endothelin and P2U-purinergic) to signal through phosphoinositidase C activation, that ET-1 can activate multiple signalling pathways in myoid cells, and that the ET-1-stimulated phosphoinositidase C responses of myoid and Sertoli cells have different regulatory characteristics.

Rat testicular myoid cells express vasopressin receptors: receptor structure, signal transduction, and developmental regulation.

Detection of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT) in the testis of several species has led to the proposal that these peptides may have a physiological role in the regulation of testicular function. Therefore, we investigated whether the contractile myoid cells of rat seminiferous tubules express functional receptors for AVP or OT and, thus, constitute a target for these hormones. This study used primary cultures of purified peritubular myoid cells derived from rats both before and after puberty. By several criteria, myoid cells prepared from adult rats expressed vasopressin receptors (VPRs). We detected specific and saturable [3H]AVP binding to a single population of sites with a Kd of 7.5 nM and a binding capacity of 145 fmol/mg protein. AVP stimulated the accumulation of inositol phosphates in a dose-dependent manner with an EC50 of 1.7 nM. Cloning and sequencing of the myoid cell VPR confirmed it to be the V1a subtype of VPR. VPR expression by myoid cells is under developmental control, as the receptors are present in the adult rat, but absent before puberty. In contrast, OT receptors were not expressed at any stage of development. Peritubular myoid cells are also responsive to endothelin-1 (ET-1), which potently stimulated phosphoinositidase-C. However, unlike AVP, the ET-1 responses were observed both before and after sexual maturity, suggesting different roles for AVP and ET-1 in the control of myoid cell function. Our data establish that the myoid cells of the adult rat seminiferous tubule are a target for AVP. This indicates an additional role for AVP in the regulation of testicular function and male fertility in the adult rat.