Characterization and regulation of yeast Ca2+-dependent phosphatidylethanolamine-phospholipase D activity.
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.
Tang
Xiaoqing
X
Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel.
Waksman
Michal
M
Ely
Yona
Y
Liscovitch
Mordechai
M
D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002794:Choline / Q000378:metabolism / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D007294:Inositol / Q000378:metabolism / Q000494:pharmacology; D008565:Membrane Proteins; D009154:Mutation; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010739:Phospholipase D / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology*
D002352:Carrier Proteins; D008565:Membrane Proteins; D010714:Phosphatidylethanolamines; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D007294:Inositol; D010739:Phospholipase D; D002794:Choline; D002118:Calcium