Abstract
In the yeast Saccharomyces cerevisiae, the mineral zinc is essential for growth and metabolism. Depletion of zinc from the growth medium of wild type cells results in changes in phospholipid metabolism, including an increase in phosphatidylinositol content (Iwanyshyn, W. M., Han, G.-S., and Carman, G. M. (2004) J. Biol. Chem. 279, 21976-21983). We examined the effects of zinc depletion on the regulation of the PIS1-encoded phosphatidylinositol synthase, the enzyme that catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol synthase activity increased when zinc was depleted from the growth medium. Analysis of a zrt1Delta zrt2Delta mutant defective in plasma membrane zinc transport indicated that the cytoplasmic levels of zinc were responsible for the regulation of phosphatidylinositol synthase. PIS1 mRNA, its encoded protein Pis1p, and the beta-galactosidase activity driven by the P(PIS1)-lacZ reporter gene were elevated in zinc-depleted cells. This indicated that the increase in phosphatidylinositol synthase activity was the result of a transcriptional mechanism. The zinc-mediated induction of the P(PIS1)-lacZ reporter gene, Pis1p, and phosphatidylinositol synthase activity was lost in zap1Delta mutant cells. These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p. Direct interaction between glutathione S-transferase (GST)-Zap1p(687-880) and a putative upstream activating sequence (UAS) zinc-responsive element in the PIS1 promoter was demonstrated by electrophoretic mobility shift assays. Mutations in the UAS zinc-responsive element in the PIS1 promoter abolished the GST-Zap1p(687-880)-DNA interaction in vitro and abolished the zinc-mediated regulation of the PIS1 gene in vivo. This work advances understanding of phospholipid synthesis regulation by zinc and the transcription control of the PIS1 gene.
Highlights
Phosphatidylinositol (PI)1 is the third most abundant phospholipid in the cellular membranes of the yeast Saccharomyces cerevisiae [1,2,3], and it is essential for the growth and metabolism of this model eukaryote (4 – 6)
These data indicated that the regulation of PIS1 gene expression by zinc depletion was mediated by the zinc-regulated transcription factor Zap1p
The yeast S. cerevisiae has the ability to cope with a variety of stress conditions by regulating the expression of enzyme activities including those involved in phospholipid synthesis [4, 27, 40, 40, 43, 66, 67]
Summary
UASINO element in the CHO1 promoter and by the transcription factors Ino2p, Ino4p, and Opi1p [40]. Our data indicated that this regulation occurred by a transcriptional mechanism that was mediated by the transcriptional activator Zap1p
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