Christophe Pical

Inositol phospholipid metabolism in arabidopsis. Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C

Phosphoinositides (PIs) constitute a minor fraction of total cellular lipids in all eukaryotic cells. They fulfill many important functions through interaction with a wide range of cellular proteins. Members of distinct inositol lipid kinase families catalyze the synthesis of these phospholipids from phosphatidylinositol. The hydrolysis of PIs involves phosphatases and isoforms of PI-specific phospholipase C. Although our knowledge of the roles played by plant PIs is clearly limited at present, there is no doubt that they are involved in many physiological processes during plant growth and development. In this review, we concentrate on inositol lipid-metabolizing enzymes from the model plant Arabidopsis for which biochemical characterization data are available, namely the inositol lipid kinases and PI-specific phospholipase Cs. The biochemical properties and structure of characterized and genome-predicted isoforms are presented and compared with those of the animal enzymes to show that the plant enzymes have some features clearly unique to this kingdom.

N‐terminal EF‐hand‐like domain is required for phosphoinositide‐specific phospholipase C activity in Arabidopsis thaliana

Phosphoinositide‐specific phospholipase Cs (PI‐PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5‐bisphosphate into the two second messengers inositol 1,4,5‐trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI‐PLCs have been identified, and the three‐dimensional structure of one rat isozyme, PLC‐δ1, determined. Plants appear to contain only one gene family encoding PI‐PLCs. The catalytic properties of plant PI‐PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI‐PLCs. All plant PI‐PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI‐PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N‐terminal domain may represent an EF‐hand domain that is required for catalytic activity but not for lipid binding.

Ca2+/phospholipid-binding (C2) domain in multiple plant proteins: novel components of the calcium-sensing apparatus.

Joachim Kopka 1, Christophe Pical 2, Alistair M. Hetherington3 and Bernd M̈uller-Röber1; Max-Planck-Institut f̈ ur Molekulare Pflanzenphysiologie (MPI-MOPP), Karl-Liebknecht-Strasse 25, Haus 20, 14476 Golm/Potsdam, Germany ( author for correspondence); 2Department of Molecular Biology and Biotechnology, University of Sheffield, PO Box 594, Sheffield S10 2UH, UK; 3Institute of Environmental and Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.

The outer membrane of plant mitochondria contains a calcium‐dependent protein kinase and multiple phosphoproteins

Highly purified mitochondria from potato (Solomon tuberosum L. cv. Bintje) tubers were subfractionated into a matrix fraction, an inner membrane fraction and an outer membrane fraction with minimal cross‐contamination. When the matrix and inner membrane fractions were incubated with [γ‐32P]ATP only one and three prominent phosphoproteins were detected after SDS‐PAGE and autoradiography, respectively. In contrast, more than 20 phosphoproteins could be labelled in the outer membrane fraction, the main ones at 12, 18, 26, 43, 58, 60, 65, 74 and 110 kDa. Only one band, at 18 kDa, was detectable when the labelling was done in the presence of EGTA. We conclude that the outer membrane of plant mitochondria contains at least one Ca2+‐dependent protein kinase and more than 20 endogenous substrates.