Transcriptional regulation of phospholipid biosynthesis is linked to fatty acid metabolism by an acyl-CoA-binding-protein-dependent mechanism in Saccharomyces cerevisiae

Abstract

In the present study, we have used DNA microarray and quantitative real-time PCR analysis to examine the transcriptional changes that occur in response to cellular depletion of the yeast acyl-CoA-binding protein, Acb1p. Depletion of Acb1p resulted in the differential expression of genes encoding proteins involved in fatty acid and phospholipid synthesis (e.g. FAS1, FAS2, ACC1, OLE1, INO1 and OPI3), glycolysis and glycerol metabolism (e.g. GPD1 and TDH1), ion transport and uptake (e.g. ITR1 and HNM1) and stress response (e.g. HSP12, DDR2 and CTT1). In the present study, we show that transcription of the INO1 gene, which encodes inositol-3-phosphate synthase, cannot be fully repressed by inositol and choline, and UAS(INO1) (inositol-sensitive upstream activating sequence)-driven transcription is enhanced in Acb1p-depleted cells. In addition, the reduction in inositol-mediated repression of INO1 transcription observed after depletion of Acb1p appeared to be independent of the transcriptional repressor, Opi1p. We also demonstrated that INO1 and OPI3 expression can be normalized in Acb1p-depleted cells by the addition of high concentrations of exogenous fatty acids, or by the overexpression of FAS1 or ACC1. Together, these findings revealed an Acb1p-dependent connection between fatty acid metabolism and transcriptional regulation of phospholipid biosynthesis in yeast. Finally, expression of an Acb1p mutant which is unable to bind acyl-CoA esters could not normalize the transcriptional changes caused by Acb1p depletion. This strongly implied that gene expression is modulated either by the Acb1p-acyl-CoA ester complex directly or by its ability to donate acyl-CoA esters to utilizing systems.

Figure 1. INO1 mRNA and protein levels during different growth conditions in Acb1p-depleted cells

Q-RT-PCR analysis of INO1 mRNA levels in wild-type (Y700, black bars) and pGAL-ACB1 (Acb1p-depleted Y700, white bars) cells grown in minimal medium (SC) supplemented with choline and/or inositol as indicated (A) or in rich medium (YPD) (C). Immunoblot analysis of protein extracts from S. cerevisiae containing Ino1p–HA. Wild-type (Y700 Ino1p–HA) and pGAL-ACB1 (Acb1p-depleted Y700 Ino1p–HA) cells were grown in SC medium supplemented with choline and/or inositol as indicated (B) or in YPD medium (D). The blots in (B) and (D) were re-probed with rabbit anti-Vac8p antibody (a gift from Professor Lois S. Weisman, Life Sciences Institute and Department of Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, U.S.A) (1:2000 dilution) to serve as loading control. Data in (A) and (C) are means±S.D. (n=3) and Western blots are representative of three independent experiments.

Figure 2. UAS_INO1-driven gene expression is increased in Acb1p-depleted cells

Wild-type (Y700, black bars) and pGAL-ACB1 (Acb1p-depleted Y700, white bars) cells were grown in YNBD medium (without inositol) or supplemented with inositol as indicated. Cells were transformed with the INO1-CYC1-lacZ plasmid and β-galactosidase activity was measured to assess the ability of UAS_INO1 elements to drive lacZ expression in wild-type and Acb1p-depleted cells. β-Galactosidase activity was defined as described in the Experimental section. Data are means±S.D. (n=3).

Figure 3. INO1 mRNA levels in wild-type and Acb1p-depleted cells with SCS2, PLD1 or HAC1 disrupted

Q-RT-PCR analysis of INO1 mRNA levels in wild-type (Y700) and pGAL-ACB1 (Acb1p-depleted Y700) cells disrupted in SCS2, PLD1 or HAC1 genes, following growth in rich (YPD) medium (A) or minimal (SC) medium supplemented with 0.2 mM inositol (B). Data are means±S.D. (n=3).

Figure 4. Acb1p-depleted cells exhibits an Opi⁻ phenotype

(A) Wild-type (Y700) and pGAL-ACB1 (Acb1p-depleted Y700) cells were tested for their ability to secrete inositol to the surrounding medium when supplemented with or without 0.5 mM palmitic acid. (B) Q-RT-PCR analysis of INO1 mRNA levels in wild-type (Y700, black bars) and pGAL-ACB1 (Acb1p-depleted Y700, white bars) cells grown in YNBD medium (without inositol) supplemented with or without 0.05% (v/v) Brij58 and 0.5 mM palmitic acid as indicated. (C) Immunoblot analysis of protein extracts from S. cerevisiae expressing Ino1p–HA. Wild-type (Y700 Ino1p–HA) and pGAL-ACB1 (Acb1p-depleted Y700 Ino1p–HA) cells were grown in YNBD medium (without inositol) supplemented with or without 0.05% (v/v) Brij 58 detergent and 0.5 mM palmitic acid as indicated. The blot was re-probed with an antibody against Vac8p to serve as a loading control. Data in (B) are means±S.D. (n=3) and the Western blot is representative of three independent experiments.

Figure 5. Fatty acid supplementation normalizes INO1 and OPI3 expression on Acb1p depletion

Q-RT-PCR analysis of INO1 (A) and OPI3 (B) mRNA levels in wild-type (Y700, black bars) and pGAL-ACB1 (Acb1p-depleted Y700, white bars) cells is shown. Cells were grown in rich medium (YPD) containing 0.05% (v/v) Brij 58 detergent in the presence or absence of 0.5 mM palmitic acid (C_16:0) or 0.5 mM palmitoleic acid (C_16:1) as indicated. Data are means±S.D. (n=3).

Figure 6. Ectopic overexpression of ACC1 and FAS1 decreases INO1 expression in Acb1p-depleted cells

(A) Q-RT-PCR analysis of INO1 mRNA levels in wild-type (Y700, black bars) and pGAL-ACB1 (Acb1p-depleted Y700, white bars) cells transformed in pairs with combinations of empty vectors or construct to be expressed as indicated. Transformants were grown in selective medium (SC −uracil −tryptophan +0.2 mM inositol). (B) Q-RT-PCR analysis of INO1 mRNA levels in wild-type (Y700, black bars) and pGAL-ACB1 (Acb1p-depleted Y700, white bars) cells transformed with either empty vector or construct to be expressed as indicated. Transformants were grown in selective medium (SC −tryptophan +0.2 mM inositol). Data are means±S.D. (n=3).

Figure 7. Acb1p affects gene expression in an acyl-CoA ester-dependent manner

Q-RT-PCR analysis of INO1 mRNA levels in wild-type (Y700), pGAL-ACB1 (Acb1p-depleted Y700), pGAL-ACB1+ACB1 (Acb1p-depleted Y700 complemented with ACB1) and pGAL-ACB1+ACB1^Y28F/^K32A (Acb1p-depleted Y700 complemented with mutant acb1) cells cultured in SC medium supplemented with 0.2 mM inositol. Data are means±S.D. (n=3).