NuA4 Lysine Acetyltransferase Complex Contributes to Phospholipid Homeostasis in Saccharomyces cerevisiae

Abstract

Actively proliferating cells constantly monitor and readjust their metabolic pathways to ensure the replenishment of phospholipids necessary for membrane biogenesis and intracellular trafficking. In Saccharomyces cerevisiae, multiple studies have suggested that the lysine acetyltransferase complex NuA4 plays a role in phospholipid homeostasis. For one, NuA4 mutants induce the expression of the inositol-3-phosphate synthase gene, INO1, which leads to excessive accumulation of inositol, a key metabolite used for phospholipid biosynthesis. Additionally, NuA4 mutants also display negative genetic interactions with sec14-1^ts , a mutant of a lipid-binding gene responsible for phospholipid remodeling of the Golgi. Here, using a combination of genetics and transcriptional profiling, we explore the connections between NuA4, inositol, and Sec14 Surprisingly, we found that NuA4 mutants did not suppress but rather exacerbated the growth defects of sec14-1^ts under inositol-depleted conditions. Transcriptome studies reveal that while loss of the NuA4 subunit EAF1 in sec14-1^ts does derepress INO1 expression, it does not derepress all inositol/choline-responsive phospholipid genes, suggesting that the impact of Eaf1 on phospholipid homeostasis extends beyond inositol biosynthesis. In fact, we find that NuA4 mutants have impaired lipid droplet levels and through genetic and chemical approaches, we determine that the genetic interaction between sec14-1^ts and NuA4 mutants potentially reflects a role for NuA4 in fatty acid biosynthesis. Altogether, our work identifies a new role for NuA4 in phospholipid homeostasis.

Keywords: FAS1/FAS2; cerulenin; inositol/choline responsive elements (ICREs); steryl esters; triacylglycerols.

Figure 1

Overview of phospholipid metabolism. Key lipid metabolic proteins mentioned in the text are indicated, transcription factors/repressors are in circles, and lipids species are indicated in black rectangles. CDP-DAG, cytidine diphosphate diacylglycerol; PA, phosphatidic acid; PI, phosphatidylinositol; PI-4-P, phosphatidylinositol-4-phosphate; TAG, triacylglycerol; SE, steryl ester.

Figure 2

NuA4 mutants exacerbate the growth defects of sec14-1^ts on inositol-depleted media. NuA4 mutants (A) eaf1Δ, (B) eaf7Δ, and (C) esa1-L254P^ts increase the growth defects of the SEC14 temperature-sensitive mutant, sec14-1^ts, under inositol-depleted conditions. Wild-type (YKB1079), sec14-1^ts (YKB3144), eaf1Δ (YKB3333), sec14-1^tseaf1Δ (YKB3935), eaf7∆ (YKB3292), sec14-1^tseaf7∆ (YKB4068), esa1-L254P^ts (YKB4236), and sec14-1^tsesa1-L254P^ts (YKB4242) cultures were grown to midlog phase in SC-inositol (+75 µM myo-inositol) prior to being diluted to an OD₆₀₀ of 0.1, and four times 10-fold serial dilutions were spotted on inositol-supplemented (75 µM myo-inositol) SC media or inositol-depleted SC media. Plates were incubated for 2 d at 30, 32, or 33.5° and images are representative of three biological replicates.

Figure 3

Transcriptional profiling of sec14-1^tseaf1Δ reveals that most ICRE-regulated phospholipid genes are not significantly derepressed. (A) Volcano plot for differentially expressed genes, sec14-1^tseaf1Δ (YKB3935) vs. sec14-1^ts (YKB3144), from RNA sequencing experiments. Differential expression levels of aligned sequences were calculated using significant thresholds set at log₂ fold change over two and FDR adjusted p-value ≤ 0.05. Significant differentially expressed genes are in red. Blue shows the significantly changed lipid metabolic genes described in (B). (B) Table listing significantly downregulated and upregulated lipid metabolic genes. (C) Relative expression levels of ICRE-containing phospholipid genes between sec14-1^tseaf1Δ and sec14-1^ts. Genes are ordered from lowest to highest fold-change expression. Genes in red are discussed further in the text.

Figure 4

Lipid droplet dynamics is impaired in EAF1 deletion mutants. (A) Wild-type (YKB1079), sec14-1^ts (YKB3144), eaf1Δ (YKB3333), and sec14-1^tseaf1Δ (YKB3935) cultures were grown to midlog phase in SC media at 30° before shifting to inositol-supplemented (75 µM myo-inositol) SC media or inositol-depleted SC media for 2 hr at 30° prior to being stained with 10 µM BODIPY 493/503 for 10 min before imaging. Images shown are midfield view of representative cells for each sample (scale bar, 10 µm). (B) Graphic display of the mean relative fluorescence density for each strain and condition. Cells were first segmented from brightfield images using a custom MATLAB script then fluorescence was measured for each cell using the outlines (see Materials and Methods for details). Mean fluorescence density was measured from 100 cells across three biological replicates relative to wild-type fluorescence in inositol-supplemented media (+SEM). Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test: **p < 0.05; n.s., not significant.

Figure 5

Inhibition of fatty acid biosynthesis causes a synthetic growth defect in sec14-1^ts. (A) Genetic inhibition of triacylglycerol and steryl ester biosynthesis does not affect the growth of sec14-1^ts. Wild-type (YKB1079), dga1Δlro1Δare1Δare2Δ (d.l.a.a.Δ; YKB4336), sec14-1^ts (YKB3144), sec14-1^tsd.l.a.a.Δ (YKB4337), eaf1Δ (YKB3333), eaf1Δd.l.a.a.Δ (YKB4338), sec14-1^tseaf1Δ (YKB3935), and sec14-1^tseaf1Δ d.l.a.a.Δ (YKB4339) cultures were grown in YPD at 30° prior to being diluted to an OD₆₀₀ of 0.1, and four times 10-fold serial dilutions were plated on inositol-supplemented (75 µM myo-inositol) SC media or inositol-depleted SC media and incubated for 2 d at 30°. (B) Inhibition of fatty acid biosynthesis by cerulenin treatment causes synthetic growth defect with sec14-1^ts. Wild-type (YKB1079), sec14-1^ts (YKB3144), eaf1Δ (YKB3333), and sec14-1^tseaf1Δ (YKB3935) cultures were grown in YPD at 30° prior to being diluted to an OD₆₀₀ of 0.1, and four times 10-fold serial dilutions were plated on YPD with indicated amounts of cerulenin for 2 d at 30, 32, 33.5, and 37°. Images are representative of three biological replicates.