Genomic analysis of the Opi- phenotype

Abstract

Most of the phospholipid biosynthetic genes of Saccharomyces cerevisiae are coordinately regulated in response to inositol and choline. Inositol affects the intracellular levels of phosphatidic acid (PA). Opi1p is a repressor of the phospholipid biosynthetic genes and specifically binds PA in the endoplasmic reticulum. In the presence of inositol, PA levels decrease, releasing Opi1p into the nucleus where it represses transcription. The opi1 mutant overproduces and excretes inositol into the growth medium in the absence of inositol and choline (Opi(-) phenotype). To better understand the mechanism of Opi1p repression, the viable yeast deletion set was screened to identify Opi(-) mutants. In total, 89 Opi(-) mutants were identified, of which 7 were previously known to have the Opi(-) phenotype. The Opi(-) mutant collection included genes with roles in phospholipid biosynthesis, transcription, protein processing/synthesis, and protein trafficking. Included in this set were all nonessential components of the NuA4 HAT complex and six proteins in the Rpd3p-Sin3p HDAC complex. It has previously been shown that defects in phosphatidylcholine synthesis (cho2 and opi3) yield the Opi(-) phenotype because of a buildup of PA. However, in this case the Opi(-) phenotype is conditional because PA can be shuttled through a salvage pathway (Kennedy pathway) by adding choline to the growth medium. Seven new mutants present in the Opi(-) collection (fun26, kex1, nup84, tps1, mrpl38, mrpl49, and opi10/yol032w) were also suppressed by choline, suggesting that these affect PC synthesis. Regulation in response to inositol is also coordinated with the unfolded protein response (UPR). Consistent with this, several Opi(-) mutants were found to affect the UPR (yhi9, ede1, and vps74).

Figure 1.

S. cerevisiae phospholipid biosynthetic pathway. CDP–DAG is the common precursor for the synthesis of phosphatidylinositol (PI) and PC. PI is made de novo from glucose-6-P but can also be synthesized from exogenously supplied inositol. PC is synthesized de novo from CDP–DAG via a series of methylation reactions carried out by the products of the CHO2 and OPI3 genes. Alternatively, PC can be made via a salvage pathway (Kennedy pathway; highlighted in yellow to facilitate discussion) when E, MME, DME, or C is supplied exogenously. Genes encoding the biosynthetic enzymes are depicted in gray boxes.

Figure 2.

Comparison of the relative percentage of genes in functional categories for the VYDS and the Opi⁻ screen. The data were obtained using the GoTermMapper program (http://db.yeastgenome.org/cgi-bin/GO/goTermMapper). Green bars reflect the percentage of genes in the Opi⁻ mutant set in each of the functional categories while red bars reflect the expected percentages of genes in the VYDS in each of the functional categories. Note that the percentage of genes in the VYDS category was modified from the on-line data set to include only nonessential genes.

Figure 3.

Opi⁻ phenotype of mutants in a HDAC chromatin modification complex. (A) A wild-type strain (BY4742) and strains containing mutations in the OPI1 gene and members of the Rpd3 HDAC (UME6, SIN3, RPD3, and PHO23) complex were grown on medium lacking inositol and choline for 72 hr at 30°. The tester strain BRS1005 was streaked perpendicularly to mutant strains and incubated at 30° for an additional 72 hr. (B) The ume1Δ and sap30Δ mutants were also tested for the Opi⁻ phenotype because these were recently found to be members of the same HDAC complex (Carrozza et al. 2005; Gavin et al. 2006). (C) The ume1Δ and sap30Δ mutants altered regulation of the INO1-lacZ reporter. Strains were grown in I−C− (derepressing; green bars) and I+C+ (repressing; red bars) conditions. Each bar is the mean of at least three independent transformants.

Figure 4.

Opi⁻ phenotype of mutants in the NuA4 chromatin-remodeling complexes. (A) Strains containing mutations in members of the NuA4 HAT complex (EAF3, EAF5, EAF7, VID21, and YAF9) were grown on medium lacking inositol and choline for 72 hr at 30°. The tester strain BRS1005 was streaked perpendicularly to mutant strains and incubated at 30° for an additional 72 hr. Refer to Figure 3A for wild-type strain control. (B) Depiction of the NuA4 complex (reproduced from Doyon and Coté 2004) listing the nonessential components. (C) Effect of the NuA4 Opi⁻ mutant on expression of an OPI1-cat reporter. Strains were grown in I−C− (derepressing; green bars) and I+C+ (repressing red bars) conditions. Each bar is the mean of at least three independent transformants.

Figure 5.

Opi⁻ mutants altered regulation of the INO1-lacZ reporter. (A) Opi⁻ mutants grown in I−C− (derepressing) conditions. The yellow bar highlights the wild-type strain. (B) Opi⁻ mutants grown in I+C+ (repressing) conditions. The yellow bar again highlights the wild-type strain. Red bars represent mutants that grew as dark blue on the X-gal plate assay (Table 2). Blue bars and open bars represent mutants that grew as medium blue and white/light blue, respectively, on the X-gal plate assay (Table 2). Each bar is the mean of at least three independent transformants. Note that 75 mutants were assayed. The remaining 14 mutants either did not grow or grew poorly under the conditions tested here. These are listed in the supplemental data at http://www.genetics.org/supplemental/.

Figure 5.

Opi⁻ mutants altered regulation of the INO1-lacZ reporter. (A) Opi⁻ mutants grown in I−C− (derepressing) conditions. The yellow bar highlights the wild-type strain. (B) Opi⁻ mutants grown in I+C+ (repressing) conditions. The yellow bar again highlights the wild-type strain. Red bars represent mutants that grew as dark blue on the X-gal plate assay (Table 2). Blue bars and open bars represent mutants that grew as medium blue and white/light blue, respectively, on the X-gal plate assay (Table 2). Each bar is the mean of at least three independent transformants. Note that 75 mutants were assayed. The remaining 14 mutants either did not grow or grew poorly under the conditions tested here. These are listed in the supplemental data at http://www.genetics.org/supplemental/.

Figure 6.

Kennedy pathway utilization suppressed Opi⁻ mutants defective in de novo synthesis of PC. The Kennedy pathway utilizes E, MME (or M), DME (or D), and C to synthesize PE, PMME, PDME, and PC, respectively. The Opi⁻ phenotype assay was performed on medium lacking inositol and supplemented with E, M, D, or C. A wild-type strain (BY4742) and an opi1Δ strain were used as controls for absence of the Opi⁻ phenotype and an unconditional Opi⁻ phenotype.

Figure 7.

Effect of Kennedy pathway on regulation of the INO1-lacZ reporter. Strains containing the INO1-lacZ reporter in pJH330 were grown in media either lacking or containing inositol ± E, MME, DME, or C. Red bars represent cells grown in I− media and green bars in I+ media. Each bar is the mean of at least three independent transformants. A wild-type strain (BY4742) and an opi1Δ strain were used as controls.

Figure 8.

Opi⁻ mutants affected the UPR. Wild type and a sample of Opi⁻ mutants were transformed with pJC104. Transformants were grown in I+C+ conditions at 30° and shifted to I+C+ or, to induce stress, I−C− and I+C+Tm+. (A) 1-hr and (B) 3-hr samples were collected and β-galactosidase activity was quantified. Each bar is the mean of three independent transformants.

Figure 9.

Schematic depicting the UPR regulatory cascade. Refer to text for description.