Systematic analysis of nuclear gene function in respiratory growth and expression of the mitochondrial genome in S. cerevisiae

Abstract

The production of metabolic energy in form of ATP by oxidative phosphorylation depends on the coordinated action of hundreds of nuclear-encoded mitochondrial proteins and a handful of proteins encoded by the mitochondrial genome (mtDNA). We used the yeast Saccharomyces cerevisiae as a model system to systematically identify the genes contributing to this process. Integration of genome-wide high-throughput growth assays with previously published large data sets allowed us to define with high confidence a set of 254 nuclear genes that are indispensable for respiratory growth. Next, we induced loss of mtDNA in the yeast deletion collection by growth on ethidium bromide-containing medium and identified twelve genes that are essential for viability in the absence of mtDNA (i.e. petite-negative). Replenishment of mtDNA by cytoduction showed that respiratory-deficient phenotypes are highly variable in many yeast mutants. Using a mitochondrial genome carrying a selectable marker, ARG8 ^m , we screened for mutants that are specifically defective in maintenance of mtDNA and mitochondrial protein synthesis. We found that up to 176 nuclear genes are required for expression of mitochondria-encoded proteins during fermentative growth. Taken together, our data provide a comprehensive picture of the molecular processes that are required for respiratory metabolism in a simple eukaryotic cell.

Keywords: mitochondria; mitochondrial DNA; oxidative phosphorylation; petite mutant; yeast.

Figure 1. FIGURE 1: Defining a set of high confidence nuclear pet mutants.

(A) Venn diagram comparing the results of four different screens ([18, 23, 25] and this study) for mutants with a pet phenotype. In each case, genome-wide collections of viable deletion mutants were analyzed and a pet phenotype was attributed to strains that were unable to grow on rich media containing glycerol as nonfermentable carbon source. (B) A pet score was derived from the four screens depicted in A by comparing the times a pet phenotype was reported for each gene to the times the deletion mutant was analyzed. See text for details. Viable deletion mutants were grouped according to their pet score and analyzed for the percentage of encoded proteins that were found in a high confidence mitochondrial proteome [8]. A detailed list containing the results from A and B can be found in Table S1. (C) Mutants with a pet score higher than 0.5, referred to as high confidence pet mutants, were manually grouped into functional categories. Lists of the genes present in each group can be found in Tables 1 and S2.

Figure 2. FIGURE 2: Contribution of mtDNA maintenance to the variability of pet phenotypes.

(A) Flow chart depicting the experimental outline. In brief, the whole deletion collection was treated with EtBr to induce loss of mtDNA. Functional [rho⁺] mtDNA was re-introduced into each strain by cytoduction. The resulting [rho⁺] deletion collection was tested for growth on rich media containing glycerol as nonfermentable carbon source. See text for details. (B) Venn diagram comparing the sets of deletion mutants that showed a pet phenotype before and after EtBr-treatment and cytoduction. The mutants were grouped into three classes: Mutants that exhibited a pet phenotype before and after cytoduction were classified as class I mutants, those that were rescued by cytoduction were grouped into class II, and mutants that were unable to grow on media with glycerol as the carbon source only after cytoduction are referred to as class III mutants. Three of the 278 pet mutants from the original deletion collection exhibited a petite-negative phenotype and were omitted from the analysis.

Figure 3. FIGURE 3: Defining the genes required for expression and maintenance of the mitochondrial genome.

(A) Flow chart depicting the outline of the experiment. A Δorf Δarg8 double mutant collection was generated using SGA technology (see methods). The mitochondrial genome was eliminated from all double mutants by treatment with EtBr. A functional mtDNA containing the ARG8^m allele was introduced into all strains by cytoduction. Mutants that were unable to grow on media lacking arginine after cytoduction were considered to have lost their mtDNA or to be unable to express the ARG8^m gene. To test for this, the resulting Δorf Δarg8 [ARG8^m] double mutant collection was crossed with a Δarg8 [rho⁰] strain and the resulting diploid strains were scored for growth on media lacking arginine. Mutants that were unable to grow were considered to suffer from mtDNA instability. See text for details. (B) The mutants that had lost their mtDNA or that were unable to express the ARG8^m gene were manually grouped into functional categories. Depicted is how often each functional group is represented among these two sets of mutants. Blue bars represent mutants that maintained the [ARG8^m] mitochondrial genome, but were unable to express Arg8^m (i.e. the genes listed in Table 3; these are the “genes for expression of mtDNA” minus “genes for maintenance of mtDNA” in panel A). Red bars represent mutants that lost the [ARG8^m] mitochondrial genome (i.e. the genes listed in Table 4; these are the “genes for maintenance of mtDNA” in panel A that could be confirmed by DAPI staining).