Detailed analysis of the turnover of polyphosphoinositides and phosphatidic acid upon activation of phospholipases C and D in Chlamydomonas cells treated with non-permeabilizing concentrations of mastoparan |
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Authors: | Teun Munnik John A J van Himbergen Bas ter Riet Franz-Josef Braun Robin F Irvine Herman van den Ende Alan Musgrave |
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Institution: | Institute for Molecular Cell Biology, section Plant Physiology, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands, NL Institut für Biochemie, Universit?t Regensburg, D-93040 Regensburg, Germany, DE Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QJ, UK, GB
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Abstract: | Treating Chlamydomonas moewusii cells with non-permeabilizing concentrations of mastoparan (1–5 μM) increased inositol 1,4,5-trisphosphate (InsP3) levels up to 20-fold in a dose-dependent manner and rapidly induced deflagellation and mating-structure activation, two
well-defined Ca2+-responses. When metabolism of the phospholipid precursors was monitored in 32Pi-labelled cells, as much as 70% of the radioactivity in phosphatidylinositol bisphosphate (PtdInsP2) was lost within 20 s. Thereafter, the 32P-label in PtdInsP2 increased to twice the control level within 10 min. A similar pattern of 32P-labelling was also exhibited by PtdInsP. An HPLC-headgroup analysis revealed that only PtdIns4P and PtdIns(4,5)P2 were involved and not the D3-phosphorylated isomers. Correlated with the increased polyphosphoinositide (PPI) turnover, there
was a massive (5- to 10-fold) increase in 32P-labelled phosphatidic acid (PtdOH) and, slightly later, an increase in its metabolic product, diacylglycerol pyrophosphate
(DGPP), reflecting the phosphorylation of the resulting diacylglycerol (DAG) and PtdOH, respectively. Mastoparan-treatment
of 32P-labelled cells in the presence of 0.2% n-butanol increased the formation of radioactive phosphatidylbutanol (PtdBut), a specific reporter of phospholipase D (PLD)
activity. This means that mastoparan activates both phospholipase C (PLC) and PLD, and thus both pathways could contribute
to the increase in PtdOH. To distinguish between them, a differential labelling strategy was applied based on the fact that
32Pi-label is slowly incorporated into structural phospholipids but rapidly incorporated into ATP. Since PLD hydrolyses a structural
lipid, radioactivity only appears slowly in PtdOHPLD (and PtdBut). In contrast, PtdOHPLC is synthesised by phosphorylation of DAG, and therefore should rapidly incorporate radioactivity. In practice, PtdOH formed
on addition of mastoparan was rapidly labelled, reflecting the specific radioactivity of the 32P]ATP pool. Based on the production of 32P]PtdBut, we estimate that about 5–17% of the PtdOH was generated through the PLD pathway, while the majority originated from
PLC activity. Together, this is the first demonstration (i) that PLC activation is correlated with increases in Ca2+, InsP3, PtdOH and DGPP, at the cost of PtdInsP and PtdInsP2, all in one and the same cell, (ii) of the characteristics of stimulated and unstimulated PPI turnover, (iii) that stimulated
turnover affects the D-4 PPI and not the 3-isomers, (iv) that PLC and PLD are activated at the same time, (v) of a simple
labelling method to discriminate between the two in terms of PtdOH production.
Received: 3 December 1997 / Accepted: 22 May 1998 |
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Keywords: | :Chlamydomonas G-protein Phospholipase Phospholipid signalling Phospholipid turnover Signal transduction |
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