The influence of phospholipid metabolism and regulatory networks on the subcellular localization of phosphatidic acid in Saccharomyces cerevisiae

Abstract

Phosphatidic acid (PA) is a signaling molecule that plays an important role in mediating yeast phospholipid biosynthesis. In cells grown in the absence of inositol, the transcriptional repressor Opi1p is localized to the endoplasmic reticulum (ER) through interactions with Scs2p via its FFAT motif (two phenylalanines in an acidic tract) and the binding with PA. Under these conditions, the UASINO‐containing phospholipid biosynthetic genes are expressed at the maximum level through activation via the Ino2p‐Ino4p activation complex. When inositol is added to the medium, the increased synthesis of phosphatidylinositol causes a rapid depletion of PA in the ER. Opi1p then translocates to the nucleus and represses the transcription of target genes (Loewen et al. 2004. Science 304, 1644 ‐ 1647). Here we present studies utilizing Opi1p reporter constructs to monitor the change in PA metabolism. Strains carrying a single genomic copy of a wild type OPI1 or a mutant OPI1FFAT fused to GFP driven by the native OPI1 promoter were constructed. These functional constructs were introduced into strains contain mutations known to perturb PA metabolism or Opi1p localization including sec14ts, scs2Δ, single mutations or in combination with pct1Δ. INO1 expression was examined to compare with Opi1p localization results and to monitor Opi1p repressor activity under changing metabolic conditions.