RESUMEN
Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca(2+) ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.
Asunto(s)
Fosfoinositido Fosfolipasa C/genética , Inmunidad de la Planta/genética , Solanum lycopersicum/genética , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/enzimología , Familia de Multigenes , Fosfoinositido Fosfolipasa C/biosíntesis , Fosfoinositido Fosfolipasa C/aislamiento & purificación , Proteínas Quinasas/genéticaRESUMEN
A recombinant metal-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) from Streptomyces antibioticus has been crystallized by the hanging-drop method with and without heavy metals. The native crystals belonged to the orthorhombic space group P222, with unit-cell parameters a=41.26, b=51.86, c=154.78â Å. The X-ray diffraction results showed significant differences in the crystal quality of samples soaked with heavy atoms. Additionally, drop pinning, which increases the surface area of the drops, was also used to improve crystal growth and quality. The combination of heavy-metal soaks and drop pinning was found to be critical for producing high-quality crystals that diffracted to 1.23â Å resolution.
Asunto(s)
Proteínas Bacterianas/química , Fosfoinositido Fosfolipasa C/química , Streptomyces antibioticus/enzimología , Proteínas Bacterianas/aislamiento & purificación , Cadmio/química , Cristalización , Electroforesis en Gel de Poliacrilamida , Iridio/química , Fosfoinositido Fosfolipasa C/aislamiento & purificación , Difracción de Rayos XRESUMEN
Phosphoinositide-specific phospholipase C (PI-PLC) activities are involved in mediating plant cell responses to environmental stimuli. Two variants of PI-PLC have been partially purified from the roots of oat seedlings; one cytosolic and one particulate. Although the cytosolic enzyme was significantly purified, the activity still co-migrated with a number of other proteins on heparin HPLC and also on size-exclusion chromatography. The partially purified PI-PLC was tested by Western blotting, and we found that actin and actin-binding proteins, profilin and tropomyosin, co-purified with cytosolic phospholipase C. After a non-ionic detergent (Triton X-100) treatment, PI-PLC activities still remained with the actin cytoskeleton. The effects of phalloidin and F-buffer confirmed this association; these conditions, which favor actin polymerization, decreased the release of PI-PLC from the cytoskeleton. The treatments of latrunculin and G-buffer, the conditions that favor actin depolymerization, increased the release of PI-PLC from the cytoskeleton. These results suggest that oat PI-PLC associates with the actin cytoskeleton.
Asunto(s)
Actinas/metabolismo , Avena/enzimología , Citoesqueleto/enzimología , Fosfoinositido Fosfolipasa C/metabolismo , Raíces de Plantas/enzimología , Citoesqueleto de Actina/metabolismo , Animales , Avena/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Bovinos , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Reacciones Cruzadas/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Detergentes/farmacología , Sueros Inmunes/farmacología , Inmunoprecipitación , Faloidina/metabolismo , Fosfoinositido Fosfolipasa C/antagonistas & inhibidores , Fosfoinositido Fosfolipasa C/aislamiento & purificación , Fosfolipasa C beta/metabolismo , Raíces de Plantas/efectos de los fármacos , Profilinas/metabolismo , Unión Proteica/efectos de los fármacos , Solubilidad/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Tiazolidinas/farmacologíaRESUMEN
Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phophatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to diacylglycerol (DAG) and inositol 1,4,5-triphosphate [Ins(1,4,5)P3]. PLCepsilon is a recently discovered isoform that has been shown to be activated by members of the Ras and Rho families of guanosine trisphosphatases (GTPases) as well as subunits of heterotrimeric G-proteins. We describe a method for expressing a truncated PLCepsilon variant as an MBP fusion protein in E. coli. Subsequently, we describe the methodology necessary to reconstitute this protein with K-Ras-4B and RhoA GTPases and measure its activation.
Asunto(s)
Baculoviridae/enzimología , Fosfoinositido Fosfolipasa C/aislamiento & purificación , Fosfoinositido Fosfolipasa C/metabolismo , Proteínas ras/aislamiento & purificación , Proteínas ras/farmacología , Proteínas de Unión al GTP rho/aislamiento & purificación , Proteínas de Unión al GTP rho/farmacología , Animales , Línea Celular , Membrana Celular/química , Sistema Libre de Células , Cromatografía de Afinidad , Cromatografía en Gel , Activación Enzimática/efectos de los fármacos , Escherichia coli/citología , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Fosfoinositido Fosfolipasa C/genética , Ratas , Eliminación de Secuencia , SolubilidadRESUMEN
Phospholipase C-eta2 (PLC-eta2) was recently identified as a novel broadly expressed phosphoinositide-hydrolyzing isozyme [Zhou, Y., et al. (2005) Biochem. J. 391, 667-676; Nakahara, M., et al. (2005) J. Biol. Chem. 280, 29128-29134]. In this study, we investigated the direct regulation of PLC-eta2 by Gbetagamma subunits of heterotrimeric G proteins. Coexpression of PLC-eta2 with Gbeta 1gamma 2, as well as with certain other Gbetagamma dimers, in COS-7 cells resulted in increases in inositol phosphate accumulation. Gbeta 1gamma 2-dependent increases in phosphoinositide hydrolysis also were observed with a truncation mutant of PLC-eta2 that lacks the long alternatively spliced carboxy-terminal domain of the isozyme. To begin to define the enzymatic properties of PLC-eta2 and its potential direct activation by Gbetagamma, a construct of PLC-eta2 encompassing the canonical domains conserved in all PLCs (PH domain through C2 domain) was purified to homogeneity after expression from a baculovirus in insect cells. Enzyme activity of purified PLC-eta2 was quantified after reconstitution with PtdIns(4,5)P 2-containing phospholipid vesicles, and values for K m (14.4 microM) and V max [12.6 micromol min (-1) (mg of protein) (-1)] were similar to activities previously observed with purified PLC-beta or PLC-epsilon isozymes. Moreover, purified Gbeta 1gamma 2 stimulated the activity of purified PLC-eta2 in a concentration-dependent manner similar to that observed with purified PLC-beta2. Activation was dependent on the presence of free Gbeta 1gamma 2 since its sequestration in the presence of Galpha i1 or GRK2-ct reversed Gbeta 1gamma 2-promoted activation. The PH domain of PLC-eta2 is not required for Gbeta 1gamma 2-mediated regulation since a purified fragment encompassing the EF-hand through C2 domains but lacking the PH domain nonetheless was activated by Gbeta 1gamma 2. Taken together, these studies illustrate that PLC-eta2 is a direct downstream effector of Gbetagamma and, therefore, of receptor-activated heterotrimeric G proteins.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Fosfoinositido Fosfolipasa C/metabolismo , Animales , Células COS , Chlorocebus aethiops , Activación Enzimática , Humanos , Fosfoinositido Fosfolipasa C/química , Fosfoinositido Fosfolipasa C/aislamiento & purificación , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismoRESUMEN
Phospholipase C (PLC) plays an important role in intracellular signal transduction by hydrolyzing phosphatidylinositol 4,5-bis-phosphate, a membrane phospholipid. Currently, thirteen mammalian PLC isozymes have been identified, which are divided into six classes on the basis of structure and mechanisms. All the PLC isozymes share common domains including catalytic X and Y domains, protein kinase C conserved region 2 (C2) domain, EF-hand motif and pleckstrin homology (PH) domain. In this study, the PLC-eta1 PH domain has been over-expressed and purified. The most undesirable feature of the protein was instability, resulting in precipitation during the purification process. With the aim of structural characterization, a solution condition was optimized using SDS-PAGE and NMR spectroscopy. A circular dichroism spectrum indicated that the PLC-eta1 PH domain mainly comprised beta-strands, which was also suggested by the 2D 1H-15N HSQC spectrum.