Phosphatidylcholine is the most abundant phospholipid in eukaryotic cells, comprising 50% of total cellular phospholipid, and thus plays a major role in cellular and organellar biogenesis. In this study, we have used both nutritional deprivation as well as a conditional temperature sensitive allele of PCT1 (CTP:phosphocholine cytidylyltransferase) coupled with an inactivated phosphatidylethanolamine methylation pathway to determine how cells respond to inactivation of phosphatidylcholine synthesis. Metabolic studies determined that phosphatidylcholine biosynthesis decreased to negligible levels within 1 h upon shift to the nonpermissive temperature for the temperature-sensitive PCT1 allele. Phosphatidylcholine mass decreased to negligible levels upon removal of choline from the medium or growth at the nonpermissive temperature, with the levels of the other major phospholipids increasing slightly. Cell growth rate visibly slowed upon cessation of phosphatidylcholine synthesis. Cells remained viable for 7-8 h after phosphatidylcholine synthesis was prevented; however, at time points beyond 8 h, viability was significantly reduced but only if the cells had been previously grown at 37 degrees C and not 25 degrees C. The inhibition of phosphatidylcholine synthesis at 37 degrees C did not alter Golgi-derived vesicle transport to the vacuole as monitored by carboxypeptidase Y processing or to the plasma membrane as determined by invertase secretion. Immunofluorescence microscopy localized Pct1p to the nucleus and nuclear membrane. Pct1p activity is regulated by Sec14p, a cytoplasm/Golgi localized phosphatidylcholine/phosphatidylinositol binding protein that regulates Golgi-derived vesicle transport partially through its ligand-dependent regulation of PCT1 derived enzyme activity. Our nuclear localization of Pct1p indicates that the regulation of Pct1p by Sec14p is indirect.
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