Model for zinc-mediated regulation of phospholipid synthesis in S. cerevisiae. The CDP-DAG and Kennedy pathways shown for the synthesis of phospholipids include the relevant steps discussed in this work. A more detailed diagram of the phospholipid synthesis pathways can be found in Refs. and . The phospholipid synthesis genes known to be regulated by zinc availability are indicated in the pathways. A, CHO1, CKI1, and EKI1 (left) and PAH1, PIS1, CKI1, and EKI1 (right) are expressed at some level when cells are grown in zinc-replete medium (depicted by numerous zinc atoms outside the nucleus). Some derepressed expression levels of CHO1, CKI1, and EKI1 (indicated by the bold arrow) are dependent on the interaction of the Ino2p-Ino4p transcriptional activation complex with the UAS_INO sequence in the promoter of these genes (, ). Under this growth condition, the repressor Opi1p is associated with the nuclear/ER membrane through interactions with PA and Scs2p (, ). B, when cytosolic zinc is limiting (depicted by a reduced number of zinc atoms outside the nucleus), the expression of the Zap1p transcriptional activator is induced (), and it binds to the UAS_ZRE sequences in the promoter of PAH1 (this work), PIS1 (), CKI1 (), and EKI1 () to increase transcription (indicated by the bold arrow). Transcription of CHO1 is attenuated in zinc-deficient cells by the interaction of Opi1p with Ino2p () (indicated by the thin arrow). Dissociation of Opi1p from the nuclear/ER membrane and its translocation into the nucleus are caused by a decrease in PA concentration due to an increase in PI synthesis via CDP-DAG () and by the induction of PA phosphatase activity (this work). Any repressive effect that Opi1p might have on the expression of CKI1 and EKI1 (not depicted in B, left side) is overcome by their Zap1p-mediated induction under this growth condition. Cho, choline; P-Cho, phosphocholine; Etn, ethanolamine; P-Etn, phosphoethanolamine.

Zinc depletion causes Zap1p-dependent induction of PA phosphatase activity encoded by PAH1 and unknown gene(s). A, wild type (strain W303-1A) and dpp1Δ lpp1Δ mutant (strain TBY1) cells were grown in the absence and presence of 1.5 μm ZnSO₄. Cell extracts were prepared and assayed for PA phosphatase activity under standard assay conditions. B, wild type (strain DY1457) and zap1Δ mutant (strain ZHY6) cells were grown in the absence and presence of 1.5 μm ZnSO₄. The cytosolic fractions were prepared and assayed for PA phosphatase activity without and with 20 mm N-ethylmaleimide (NEM). Each data point represents the average of triplicate enzyme determinations from two independent experiments ±S.D. (error bars).

Depletion of cytosolic zinc causes Zap1p-mediated induction of P_PAH1-lacZ reporter gene activity. A, wild type (strain W303-1A) cells bearing the P_PAH1-lacZ reporter plasmid pFP1 were grown in the presence of the indicated concentrations of ZnSO₄. B, wild type (strain DY1457), zrt1Δ zrt2Δ mutant (strain ZHY3), and zap1Δ mutant (strain ZHY6) cells bearing the P_PAH1-lacZ plasmid were grown in the absence and presence of 1.5 μm ZnSO₄. Cell extracts were prepared and assayed for β-galactosidase activity. Each data point represents the average of triplicate enzyme determinations from a minimum of two independent experiments ±S.D. (error bars). The differences in the β-galactosidase activities of the wild type controls in A and B were due to strain differences.

Interactions of GST-Zap1p(687–880) with putative UAS_ZRE sequences in PAH1 promoter. A, the locations and sequences of the putative UAS_ZRE sequences in the PAH1 promoter. B, samples (1 pmol) of radiolabeled double-stranded synthetic oligonucleotides (2.0 × 10⁵ cpm/pmol) with sequences for ZRE1, ZRE2, and ZRE3 in the PAH1 promoter were incubated with 0, 0.15, 0.3, and 0.5 μg of purified recombinant GST-Zap1p(687–880). C, GST-Zap1p(687–880) (0. 5 μg) was incubated with 0, 25, 50, and 100 pmol of unlabeled oligonucleotide with sequences for ZRE1, ZRE2, and ZRE3, respectively. Interactions of GST-Zap1p(687–880) with the labeled oligonucleotides were determined by electrophoretic mobility shift assays using 6% polyacrylamide gels. The data shown are representative of three independent experiments. The arrow indicates the position of the GST-Zap1p(687–880)-ZRE complex.

pah1Δ mutation causes derepression of PS synthase activity and ablates zinc-mediated regulation of enzyme. Wild type (strain W303-1A), dpp1Δ lpp1Δ mutant (strain TBY1), and pah1Δ mutant (strain GHY57) cells were grown in the absence and presence of 1.5 μm ZnSO₄. Cell extracts were prepared and assayed for PS synthase activity. Each data point represents the average of triplicate enzyme determinations from two independent experiments ±S.D. (error bars).

pah1Δ mutation causes derepression of choline kinase activity but inhibits synthesis of PC via Kennedy pathway when cells are deficient of zinc. Wild type (strain W303-1A) and pah1Δ mutant (strain GHY57) cells were grown in the absence and presence of 1.5 μm ZnSO₄. A, cell extracts were prepared and assayed for choline kinase activity. Each data point represents the average of triplicate enzyme determinations from two independent experiments ±S.D. B, the cultures were incubated with [methyl-¹⁴C]choline (0.2 μCi/ml) to uniformly label the Kennedy pathway intermediates and PC. The water-soluble intermediates and PC were extracted and analyzed separately by TLC. The ¹⁴C-labeled compounds were visualized by phosphorimaging, and their amounts in cpm were determined from a standard curve using ImageQuant software. The values reported are the average of three separate experiments ±S.D. (error bars). Cho, choline; P-Cho, phosphocholine.

Neutral lipid composition is affected in pah1Δ mutant cells deficient of zinc. Wild type (strain W303-1A) and pah1Δ mutant (strain GHY57) cells were grown in the absence and presence of 1.5 μm ZnSO₄. The cultures were incubated with [2-¹⁴C]acetate (1 μCi/ml) to uniformly label cellular lipids. Lipids were extracted and separated by one-dimensional TLC. The ¹⁴C-labeled lipids were visualized by phosphorimaging and quantified by ImageQuant analysis. The percentages shown for the individual lipids were normalized to the total ¹⁴C-labeled chloroform-soluble fraction. The values reported are the average of three separate experiments ±S.D. (error bars). ErgE, ergosterol esters; Erg, ergosterol.