SPO14 Separation-of-Function Mutations Define Unique Roles for Phospholipase D in Secretion and Cellular Differentiation in Saccharomyces cerevisiae

Abstract 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.

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Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity

Biochemistry and Cell Biology ◽

10.1139/o92-006 ◽

1992 ◽

Vol 70 (1) ◽

pp. 43-48 ◽

Cited By ~ 3

Author(s):

S. S. Ghosh ◽

Richard C. Franson

Keyword(s):

Escherichia Coli ◽

Phosphatidic Acid ◽

Phospholipase D ◽

Phospholipase A ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

E Coli ◽

Streptomyces Chromofuscus ◽

Hydrolysis Of

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0–8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 μM) or propanolol (IC50 = 180 μM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.Key words: Escherichia coli, substrate, phospholipase D, Streptomyces chromofuscus, sodium fluoride, propranolol.

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Activation of phospholipase D by α-thrombin or epidermal growth factor contributes to the formation of phosphatidic acid, but not to observed increases in 1,2-diacylglycerol

Biochemical Journal ◽

10.1042/bj2850395 ◽

1992 ◽

Vol 285 (2) ◽

pp. 395-400 ◽

Cited By ~ 25

Author(s):

T M Wright ◽

S Willenberger ◽

D M Raben

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Phosphatidic Acid ◽

Phospholipase D ◽

Myristic Acid ◽

Transient Activation ◽

Lipid Species ◽

Epidermal Growth

The receptor-mediated activation of a phosphatidylcholine-hydrolysing phospholipase D (PLD) has recently been described. We investigated the effect of alpha-thrombin and epidermal growth factor (EGF) on cellular PLD activity in order to determine the role of this enzyme in mitogen-induced increases in phosphatidic acid and sn-1,2-diacylglycerol. In the presence of ethanol, stimulation of [3H]myristic acid-labelled quiescent IIC9 cells with alpha-thrombin or EGF resulted in a rapid increase in radiolabelled phosphatidyl-ethanol which reached a plateau at 1 min, indicating the rapid and transient activation of PLD. We observed a concomitant decrease in the mitogen-stimulated increase of radiolabelled phosphatidic acid. In contrast, ethanol did not significantly effect the elevation of sn-1,2-diacylglycerol levels stimulated by alpha-thrombin or EGF as determined by measurement of sn-1,2-diacylglycerol mass or the appearance of [3H]1,2-diacylglycerol. A novel lipid, detected by two-dimensional t.l.c. analysis, was generated in [3H]myristic acid-labelled cells stimulated with alpha-thrombin, but not EGF, in the presence of ethanol. Treatment in vitro of cellular lipids isolated from [3H]myristic acid-labelled cultures with PLD in the presence of ethanol also resulted in the generation of this novel lipid species, supporting the role of this enzyme in its production. These data indicate that in quiescent IIC9 cells: (a) alpha-thrombin or EGF rapidly and transiently activates a PLD; (b) although this activation is responsible for part of the mitogen-induced increases in phosphatidic acid, it does not contribute to induced increases in sn-1,2-diacylglycerol; and (c) activation of this enzyme appears to be involved in the formation of a novel lipid generated in response to alpha-thrombin, but not EGF, in IIC9 fibroblasts.

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Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

Biochemical Journal ◽

10.1042/bj3600707 ◽

2001 ◽

Vol 360 (3) ◽

pp. 707-715 ◽

Cited By ~ 35

Author(s):

Trevor R. PETTITT ◽

Mark McDERMOTT ◽

Khalid M. SAQIB ◽

Neil SHIMWELL ◽

Michael J. O. WAKELAM

Keyword(s):

Liquid Chromatography ◽

Phosphatidic Acid ◽

Phospholipase D ◽

Mammalian Cells ◽

Inducible Promoter ◽

In Vitro Assays ◽

Protein Levels ◽

Intracellular Messenger

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

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Suppressor Analysis of the Saccharomyces cerevisiae Gene REC104 Reveals a Genetic Interaction With REC102

Genetics ◽

10.1093/genetics/151.4.1261 ◽

1999 ◽

Vol 151 (4) ◽

pp. 1261-1272 ◽

Cited By ~ 1

Author(s):

Laura Salem ◽

Natalie Walter ◽

Robert Malone

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Recombination ◽

Null Allele ◽

Genetic Interaction ◽

In Vitro Mutagenesis ◽

Conditional Mutations ◽

Suppressor Analysis

Abstract REC104 is a gene required for the initiation of meiotic recombination in Saccharomyces cerevisiae. To better understand the role of REC104 in meiosis, we used an in vitro mutagenesis technique to create a set of temperature-conditional mutations in REC104 and used one ts allele (rec104-8) in a screen for highcopy suppressors. An increased dosage of the early exchange gene REC102 was found to suppress the conditional recombinational reduction in rec104-8 as well as in several other conditional rec104 alleles. However, no suppression was observed for a null allele of REC104, indicating that the suppression by REC102 is not “bypass” suppression. Overexpression of the early meiotic genes REC114, RAD50, HOP1, and RED1 fails to suppress any of the rec104 conditional alleles, indicating that the suppression might be specific to REC102.

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The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3197-3207.1994 ◽

1994 ◽

Vol 14 (5) ◽

pp. 3197-3207

Author(s):

P R Caron ◽

P Watt ◽

J C Wang

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Localization ◽

Topoisomerase Ii ◽

Dna Topoisomerase Ii ◽

Dna Topoisomerase ◽

Mutant Proteins ◽

Terminal Domain ◽

Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

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Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

The Journal of Cell Biology ◽

10.1083/jcb.200405018 ◽

2004 ◽

Vol 167 (1) ◽

pp. 75-85 ◽

Cited By ~ 79

Author(s):

Brenton L. Scott ◽

Jeffrey S. Van Komen ◽

Hassan Irshad ◽

Song Liu ◽

Kirilee A. Wilson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Fusion ◽

Membrane Trafficking ◽

Snare Complex ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Bud Neck ◽

Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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Propranolol Inhibits Hyphal Development in Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.11.3617-3620.2002 ◽

2002 ◽

Vol 46 (11) ◽

pp. 3617-3620 ◽

Cited By ~ 15

Author(s):

Carol A. Baker ◽

Kevin Desrosiers ◽

Joseph W. Dolan

Keyword(s):

Growth Rate ◽

Candida Albicans ◽

Phosphatidic Acid ◽

Rate Data ◽

Dimorphic Transition ◽

Hyphal Development ◽

Germ Tubes ◽

Hydrolysis Of

ABSTRACT Propranolol was used to investigate the role of phosphatidic acid (PA) and diacylglycerol in the dimorphic transition in Candida albicans. Propranolol was able to inhibit the appearance of germ tubes without decreasing growth rate. Data suggest that inhibition of morphogenesis may be due to binding by propranolol of PA derived from PLD1 hydrolysis of phosphatidylcholine.

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MicroRNAs in the tumour microenvironment: big role for small players

Endocrine Related Cancer ◽

10.1530/erc-13-0119 ◽

2013 ◽

Vol 20 (5) ◽

pp. R257-R267 ◽

Cited By ~ 37

Author(s):

Patsy Soon ◽

Hippokratis Kiaris

Keyword(s):

Clinical Characteristics ◽

Experimental Studies ◽

Anticancer Therapy ◽

Tumour Microenvironment ◽

Regulatory Role ◽

Tumour Stroma ◽

Physiological Processes ◽

Non Coding Rnas

MicroRNAs (miRNAs) represent a class of small non-coding RNAs with an important regulatory role in various physiological processes as well as in several pathologies including cancers. It is noteworthy that recent evidence suggests that the regulatory role of miRNAs during carcinogenesis is not limited to the cancer cells but they are also implicated in the activation of tumour stroma and its transition into a cancer-associated state. Results from experimental studies involving cells culturedin vitroand mice bearing experimental tumours, corroborated by profiling of clinical cancers for miRNA expression, underline this role and identify miRNAs as a potent regulator of the crosstalk between cancer and stroma cells. Considering the fundamental role of the tumour microenvironment in determining both the clinical characteristics of the disease and the efficacy of anticancer therapy, miRNAs emerge as an attractive target bearing important prognostic and therapeutic significance during carcinogenesis. In this article, we will review the available results that underline the role of miRNAs in tumour stroma biology and emphasise their potential value as tools for the management of the disease.

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Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes

The Journal of Cell Biology ◽

10.1083/jcb.200205056 ◽

2002 ◽

Vol 159 (6) ◽

pp. 1039-1049 ◽

Cited By ~ 77

Author(s):

Vicki A. Sciorra ◽

Simon A. Rudge ◽

Jiyao Wang ◽

Stuart McLaughlin ◽

JoAnne Engebrecht ◽

...

Keyword(s):

Phospholipase D ◽

Membrane Trafficking ◽

Dual Role ◽

Biological Functions ◽

Ph Domain ◽

Catalytically Active ◽

Acidic Phospholipids ◽

Stimulation Of

Phospholipase D (PLD) generates lipid signals that coordinate membrane trafficking with cellular signaling. PLD activity in vitro and in vivo is dependent on phosphoinositides with a vicinal 4,5-phosphate pair. Yeast and mammalian PLDs contain an NH2-terminal pleckstrin homology (PH) domain that has been speculated to specify both subcellular localization and regulation of PLD activity through interaction with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2). We report that mutation of the PH domains of yeast and mammalian PLD enzymes generates catalytically active PI(4,5)P2-regulated enzymes with impaired biological functions. Disruption of the PH domain of mammalian PLD2 results in relocalization of the protein from the PI(4,5)P2-containing plasma membrane to endosomes. As a result of this mislocalization, mutations within the PH domain render the protein unresponsive to activation in vivo. Furthermore, the integrity of the PH domain is vital for yeast PLD function in both meiosis and secretion. Binding of PLD2 to model membranes is enhanced by acidic phospholipids. Studies with PLD2-derived peptides suggest that this binding involves a previously identified polybasic motif that mediates activation of the enzyme by PI(4,5)P2. By comparison, the PLD2 PH domain binds PI(4,5)P2 with lower affinity but sufficient selectivity to function in concert with the polybasic motif to target the protein to PI(4,5)P2-rich membranes. Phosphoinositides therefore have a dual role in PLD regulation: membrane targeting mediated by the PH domain and stimulation of catalysis mediated by the polybasic motif.

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GABAA receptor currents in the dorsal motor nucleus of the vagus in females: influence of ovarian cycle and 5α-reductase inhibition

Journal of Neurophysiology ◽

10.1152/jn.00039.2019 ◽

2019 ◽

Vol 122 (5) ◽

pp. 2130-2141

Author(s):

Erica L. Littlejohn ◽

Liliana Espinoza ◽

Monica M. Lopez ◽

Bret N. Smith ◽

Carie R. Boychuk

Keyword(s):

Sex Differences ◽

Motor Neurons ◽

Ovarian Cycle ◽

Receptor Activity ◽

Motor Nucleus ◽

Gabaergic Neurotransmission ◽

Physiological Processes ◽

Dorsal Motor Nucleus

The dorsal motor nucleus of the vagus (DMV) contains the preganglionic motor neurons important in the regulation of glucose homeostasis and gastrointestinal function. Despite the role of sex in the regulation of these processes, few studies examine the role of sex and/or ovarian cycle in the regulation of synaptic neurotransmission to the DMV. Since GABAergic neurotransmission is critical to normal DMV function, the present study used in vitro whole cell patch-clamping to investigate whether sex differences exist in GABAergic neurotransmission to DMV neurons. It additionally investigated whether the ovarian cycle plays a role in those sex differences. The frequency of phasic GABAA receptor-mediated inhibitory postsynaptic currents in DMV neurons from females was lower compared with males, and this effect was TTX sensitive and abolished by ovariectomy (OVX). Amplitudes of GABAergic currents (both phasic and tonic) were not different. However, females demonstrated significantly more variability in the amplitude of both phasic and tonic GABAA receptor currents. This difference was eliminated by OVX in females, suggesting that these differences were related to reproductive hormone levels. This was confirmed for GABAergic tonic currents by comparing females in two ovarian stages, estrus versus diestrus. Female mice in diestrus had larger tonic current amplitudes compared with those in estrus, and this increase was abolished after administration of a 5α-reductase inhibitor but not modulation of estrogen. Taken together, these findings demonstrate that DMV neurons undergo GABAA receptor activity plasticity as a function of sex and/or sex steroids. NEW & NOTEWORTHY Results show that GABAergic signaling in dorsal vagal motor neurons (DMV) demonstrates sex differences and fluctuates across the ovarian cycle in females. These findings are the first to demonstrate that female GABAA receptor activity in this brain region is modulated by 5α-reductase-dependent hormones. Since DMV activity is critical to both glucose and gastrointestinal homeostasis, these results suggest that sex hormones, including those synthesized by 5α-reductase, contribute to visceral, autonomic function related to these physiological processes.

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