Identification of a novel, Ca²⁺‐dependent phospholipase D with preference for phosphatidylserine and phosphatidylethanolamine in Saccharomyces cerevisiae

Abstract

A membrane-bound phospholipase D (PLD) fromSaccharomyces cerevisiae was solubilized from mitochondrial and plasma membranes and partially purified. The enzyme has an apparent molecular weight of approximately 60 kDa, is strictly Ca2+-dependent and preferentially hydrolyses phosphatidylserine and phosphatidylethanolamine. Enzyme activity is significantly increased in membranes from cells grown on a nonfermentable carbon source. The CaZ+-dependent PLD is distinct from PLD encoded by the SPO141PLDI gene. The 195 kDa SPO141PLDI gene product is specific for PtdCho, Ca 2+-independent and is activated by PIP2. Furthermore, Pldlp has transphosphatidylation activity in the presence of ethanol and thus resembles the prototypic PLD activity found in mammalian cells and plants. In contrast, the Ca2+-dependent PLD described here is not affected by PIP2 and does not catalyze transphosphatidylation. Thus, the Ca2+-dependent PLD characterized in this study appears to be a member of a novel family of phospholipases D.

ADP-Ribosylation Factors Do Not Activate Yeast Phospholipase Ds but Are Required for Sporulation

“…Yeast also contain a second PLD activity, termed ScPld2 (Mayr et al, 1996;Waksman et al, 1997). In contrast to Spo14 Waksman et al, 1996), PLD1 (Hammond et al, 1995, and PLD2 (Colley et al, 1997;Kodaki and Yamashita, 1997), ScPld2 does not require PIP 2 for catalysis and does not perform transphosphatidylation (Mayr et al, 1996;Waksman et al, 1997).…”

“…The function of this second activity is unknown; however, it is clear that this activity cannot substitute for Spo14 in meiosis. Furthermore, the biochemical properties of this second activity are distinct from Spo14; ScPld2 preferentially hydrolyses phosphatidylserine and phosphatidylethanolamine (Mayr et al, 1996), requires calcium for catalytic activity, but not PIP 2 , and does not catalyze transphosphatidylation in the presence of alcohols (Mayr et al, 1996;Waksman et al, 1997). To determine whether Arf1 or Arf2 activates ScPld2, we assayed total cell lysates for PE-PLD activity in spo14 deletion, spo14 arf1, spo14 arf2, and spo14 deletion strains overexpressing dominant activating ([Q71L]Arf1) or inactivating ([N126I]Arf1) proteins .…”

“…cerevisiae contain a second PLD activity, termed ScPld2, which is easily assayed in spo14 deletion strains (Mayr et al, 1996;Waksman et al, 1997). The function of this second activity is unknown; however, it is clear that this activity cannot substitute for Spo14 in meiosis.…”

An Iron-dependent Bacterial Phospholipase D Reminiscent of Purple Acid Phosphatases

“…The involvement of two HKD motifs in the formation of the active site was confirmed by the first PLD crystal structure (12). The only known examples of PLDs not containing an HKD motif are GPI-PLD (14), phosphatidylethanol-specific PLD (PE-PLD) (15,16) and autotaxin (17,18), a known ectonucleotide phosphatase/phosphodiesterase recently characterized as a lysophospholipase D (19). In Ca 2ϩ -dependent PLDs, no activity is detected in the absence of this metal ion, which is believed to be an essential cofactor in substrate binding and hydrolysis (20).…”

“…When physiologically relevant phospholipids are used as a substrate, addition of Co 2ϩ induces less than a 2-fold activation. Other reported examples of non-HKD PLD enzymes include GPI-PLD (14) and PE-PLD (15,16). While the former requires metal ions for activity (Ca 2ϩ and Zn 2ϩ ), none of these enzymes have been reported to require iron.…”