Details

Autor(en) / Beteiligte

• Munnik, T
• Himbergen, J.A.J. van
• Riet, B. ter
• Braun, F.J
• Irvine, R.F
• Ende, H. van den
• Musgrave, A

Titel

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

Ist Teil von

• Planta, 1998-12, Vol.207 (1), p.133-145

Ort / Verlag

Berlin: Springer-Verlag

Erscheinungsjahr

1998

Link zum Volltext

Quelle

SpringerLINK Contemporary (Konsortium Baden-Württemberg)

Beschreibungen/Notizen

• Treating Chlamydomonas moewusii cells with non-permeabilizing concentrations of mastoparan (1-5 micromolar) 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 32P(i)-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 mm. 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 32P(i)-label is slowly incorporated into structural phospholipids but rapidly incorporated into ATP. Since PLD hydrolyses a structural lipid, radioactivity only appears slowly in PtdOH(PLD) (and PtdBut). In contrast, PtdOH(PLC) is synthesised by phosphorylation of DAG, and therefore should rapidly incorporate radioactivity. In practice, Ptd(OH) 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.