Abstract
The SEC14 gene encodes a phosphatidylinositol/phosphatidylcholine transfer protein essential for secretion and growth in yeast (1). Mutations (cki1, cct1, and cpt1) in the CDP-choline pathway for phosphatidylcholine synthesis suppress the sec14 growth defect (2), permitting sec14(ts) cki1, sec14(ts) cct1, and sec14(ts) cpt1 strains to grow at the sec14(ts) restrictive temperature. Previously, we reported that these double mutant strains also excrete the phospholipid metabolites, choline and inositol (3). We now report that these choline and inositol excretion phenotypes are eliminated when the SPO14 (PLD1) gene encoding phospholipase D1 is deleted. In contrast to sec14(ts) cki1 strains, sec14(ts) cki1 pld1 strains are not viable at the sec14(ts) restrictive temperature and exhibit a pattern of invertase secretion comparable with sec14(ts) strains. Thus, the PLD1 gene product appears to play an essential role in the suppression of the sec14(ts) defect by CDP-choline pathway mutations, indicating a role for phospholipase D1 in growth and secretion. Furthermore, sec14(ts) strains exhibit elevated Ca2+-independent, phophatidylinositol 4,5-bisphosphate-stimulated phospholipase D activity. We also propose that phospholipase D1-mediated phosphatidylcholine turnover generates a signal that activates transcription of INO1, the structural gene for inositol 1-phosphate synthase.
Highlights
All sec14ts cki1 pld1 strains (11 independent segregants) were unable to grow at 37 °C and lacked the OpiϪ and OpcϪ phenotypes at 30 °C (Fig. 1)
In 35 tetrads analyzed from a cross of a sec14ts pld1 to a sec14ts cct1 strain, all sec14ts cct1 strains tested exhibited OpiϪ and OpcϪ phenotypes at 30 °C, whereas all sec14ts cct1 pld1 strains tested were unable to grow at 37 °C and lacked OpiϪ and OpcϪ phenotypes at 30 °C
We have demonstrated that the high level of PC turnover, producing the choline excretion phenotype previously documented in sec14ts cki1 cells growing at 37 °C (3), requires functional Pld1p (Fig. 1)
Summary
Yeast Strains—A disruption of the PLD1 (SPO14) gene (pld1::URA3), as described by Waksman et al (19) was made in a sec14ts cki strain (genotype Mata ura his lys sec14 –3ts cki1::HIS3) Assay for OpcϪ and OpiϪ—Strains were grown on plates containing complete synthetic medium lacking inositol and choline (IϪCϪ medium (3)) at 30 °C. Phospholipase D Assay—Yeast strains were grown to mid-logarithmic phase in IϪCϪ medium at 30 °C. Invertase Assay—Strains were grown in 1% yeast extract, 2% Bactopeptone, 3% glucose (YEPD) medium at 25 °C until mid-logarithmic phase of growth. At this time, they were shifted to low glucose YEP medium for 1 h at 25 or 37 °C. Lipid Analysis—Strains were grown in the presence of H332PO4 (10 Ci/ml) to uniform labeling at 30 °C in IϪCϪ medium and were
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call