An unconventional phospholipase D (PLD) activity was identified recently in Saccharomyces cerevisiae which is Ca2+-dependent, preferentially hydrolyses phosphatidylethanolamine (PtdEtn) and phosphatidylserine and does not catalyse a transphosphatidylation with primary short-chain alcohols. We have characterized the cytosolic and membrane-bound forms of the yeast PtdEtn-PLD and examined the regulation of its activity under certain growth, nutritional and stress conditions. Both forms of PtdEtn-PLD activity were similarly activated by Ca2+ ions in a biphasic manner. Likewise, other divalent cations affected both cytosolic and membrane-bound forms to the same extent. The yeast PtdEtn-PLD activity was found to interact with immobilized PtdEtn in a Ca2+-dependent manner. The partially purified cytosolic form and the salt-extracted membrane-bound form of yeast PtdEtn-PLD exhibited a similar elution pattern on size-exclusion chromatography, coeluting as low apparent molecular weight peaks. PtdEtn-PLD activity was stimulated, along with Spo14p/Pld1p activity, upon dilution of stationary phase cultures in glucose, acetate and galactose media, but PtdEtn-PLD activation was less pronounced. Interestingly, PtdEtn-PLD activity was found to be elevated by approximately 40% in sec14ts mutants at the restrictive temperature, whereas in other sec mutants it remained unaffected. The activity of PtdEtn-PLD was reduced by 30-40% upon addition to the medium of inositol (75 micro m) in either wild-type yeast or spo14Delta mutants and this effect was seen regardless of the presence of choline, suggesting that transcription of the PtdEtn-PLD gene is down-regulated by inositol. Finally, exposure of yeast cells to H2O2 resulted in a transient increase in PtdEtn-PLD activity followed by a profound, nearly 90% decrease in activity. In conclusion, our results indicate that yeast PtdEtn-PLD activity is highly regulated: the enzyme is acutely activated upon entry into the cell cycle and following inactivation of sec14ts, and is inhibited under oxidative stress conditions. The implications of these findings are discussed.

• PMID: 12153579
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Tang X, Waksman M, Ely Y, Liscovitch M

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