Genome-Wide Functional Profiling Identifies Genes and Processes Important for Zinc-Limited Growth of Saccharomyces cerevisiae

Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. To discover genes and processes in yeast that are required for growth when zinc is limiting, we used genome-wide functional profiling. Mixed pools of ∼4,600 deletion mutants were inoculated into zinc-replete and zinc-limiting media. These cells were grown for several generations, and the prevalence of each mutant in the pool was then determined by microarray analysis. As a result, we identified more than 400 different genes required for optimal growth under zinc-limiting conditions. Among these were several targets of the Zap1 zinc-responsive transcription factor. Their importance is consistent with their up-regulation by Zap1 in low zinc. We also identified genes that implicate Zap1-independent processes as important. These include endoplasmic reticulum function, oxidative stress resistance, vesicular trafficking, peroxisome biogenesis, and chromatin modification. Our studies also indicated the critical role of macroautophagy in low zinc growth. Finally, as a result of our analysis, we discovered a previously unknown role for the ICE2 gene in maintaining ER zinc homeostasis. Thus, functional profiling has provided many new insights into genes and processes that are needed for cells to thrive under the stress of zinc deficiency

Flow cytometry analysis of low zinc resistant mutants.

a<p>Significance was defined as having a p-value less than 0.05 comparing the mutant prevalence after 15 generations growth in low versus high zinc; NS = not significant.</p

Effects of zinc status on sensitivity to an ER stress inducer.

<p>Wild-type (BY4743) cells were inoculated into zinc-replete (LZM+100 µM ZnCl₂, +Zn) or zinc-limiting (LZM+1 µM ZnCl₂, −Zn) media containing a range of tunicamycin concentrations and grown overnight prior to measuring the culture optical densities at 600 nm (OD₆₀₀). Hypersensitivity to tunicamycin was observed for zinc-limited cells at 0.1, 0.25, and 0.5 mM tunicamcyin. Data presented are the averages of triplicate cultures for each condition and the error bars indicate ±1 S.D. For most points, the symbols obscure the error bars.</p

Functional profiling analysis.

<p>A) Zinc limitation in LZM+1 µM ZnCl₂ (LZM1) results in decreased growth of wild-type cells relative to replete LZM+100 µM ZnCl₂ (LZM100). A <i>tsa1</i>Δ mutant shows increased sensitivity to limiting zinc relative to wild type. B) Numbers of low zinc sensitive and resistant deletion strains identified by differential strain sensitivity analysis (DSSA). The number of significantly affected strains identified was greater after more generations of growth. C) Venn diagrams showing the number of genes whose mutants showed growth effects after five and fifteen generations. The degree of overlap is also indicated.</p

Macroautophagy is required for zinc-limited growth but not cargo-specific autophagy pathways.

a<p>Significance was defined as having a p-value less than 0.05 comparing the mutant prevalence after 15 generations growth in low versus high zinc; NS = not significant.</p

Enrichment among low zinc-sensitive mutants by biological process, cellular component, functional classification, and subcellular localization.

a<p>k = number of genes of specific category identified by screen that when deleted increase sensitivity to low zinc.</p>b<p>f = number of genes in specific GO/MIPS category.</p

A possible role for <i>ICE2</i> in ER zinc homeostasis.

<p>A) Confirmation of the <i>ice2Δ</i> low zinc growth defect. Wild type (BY4743, <i>filled columns</i>) and <i>ice2Δ</i> (BY4743 <i>ice2Δ</i>, <i>open columns</i>) cells were inoculated into LZM supplemented with either 1 or 3 µM ZnCl₂ and grown overnight prior to measuring the culture optical densities at 600 nm (OD₆₀₀). B) Loss of Ice2 causes a zinc-suppressible hyper-induction of the unfolded protein response (UPR). Wild type (BY4743) and homozygous <i>ice2Δ</i> mutant (BY4743 <i>ice2Δ</i>) cells were transformed with the UPRE-lacZ reporter pMCZ-Y and inoculated into low zinc medium (LZM) supplemented with 0.3, 1, 3 or 10 µM ZnCl₂. These cells were then grown overnight prior to measuring β–galactosidase activity. C) Loss of Ice2 exacerbates the zinc-suppressible hyper-induction of the UPR in <i>msc2Δ zrg17Δ zrc1Δ cot1Δ</i> quadruple mutants. JSY5 (<i>msc2Δ zrg17Δ zrc1Δ cot1Δ</i>) and JSY5 <i>ice2Δ</i> (<i>msc2Δ zrg17Δ zrc1Δ cot1Δ ice2Δ</i>) cells were transformed with the UPRE-lacZ reporter pMCZ-Y and inoculated into low zinc medium (LZM) supplemented with 1, 3, 10 or 100 µM ZnCl₂. These cells were then grown overnight prior to measuring β–galactosidase activity. The wild-type strain used was the isogenic CM100 strain. D) Zinc treatment does not inhibit the UPR induction in response to tunicamycin. Wild-type BY4743 cells bearing the UPRE-lacZ reporter were grown to exponential phase in LZM supplemented with the indicated concentration of zinc, then treated for 2 hours with 2 µg/ml tunicamycin prior to β–galactosidase activity assay. Data presented are the averages of triplicate cultures for each condition and the error bars indicate ±1 S.D.</p

Fitness data for all significantly affected sensitive strains identified from this study were mapped onto the <i>S. cerevisiae</i> BioGRID interaction dataset using Cytoscape.

<p>The fitness scores (the difference in the mean of the log₂ hybridization signal between LZM+1 µM ZnCl₂ and LZM+100 µM ZnCl₂) of these sensitive strains were then used to identify and create a smaller sub-network (283 genes) containing the sensitive genes and the non-sensitive and essential genes that link them through known genetic and physical interactions. The sub-network was then assessed for significant overrepresentation of Gene Ontology (GO) Cellular Component categories. These categories were visualized as a linked network. Node color of categories indicates the significance of representation (white = not identified as significant) and node size indicates the number of genes identified present in each category. Edge arrows indicate hierarchy of GO terms. For clarity, only GO Cellular Component categories with a p-value<0.0005 are shown. A separate GO enrichment assessment identified overrepresentation of all GO categories in the sub-network. This analysis was used to generate visual representations of the GO processes and cellular components identified showing the genes involved in these processes. In these cases, node color indicates the sensitivity of each deletion strain in our study (fitness score). The edge color defines the interaction type between nodes (from the BioGRID database).</p

Representative mutants showing growth defects in low zinc.

<p>Yeast pools were grown in zinc-replete (LZM+100 µM ZnCl₂) and zinc-limiting (LZM+1 µM ZnCl₂) media for either five or fifteen generations. The yeast ORFs/genes correspond to representative deletion strains that exhibited a significant change in growth in low zinc when compared to zinc-replete conditions (q<0.05). Numeric values are fitness scores (log₂ ratios); a negative value indicates a growth defect of the mutant in low zinc. Empty cells in the table indicate that any differences observed were not significant at that generation point.</p>a<p>Average fitness scores for representative sensitive strains in four replicate experiments after 5 generations of growth.</p>b<p>Average fitness scores for representative sensitive strains in four replicate experiments after 15 generations of growth.</p

Analysis of low zinc growth by flow cytometry.

<p>Untagged wild-type BY4743 (panels A, B), <i>gal2Δ</i> (panels C, D), and <i>tsa1Δ</i> (panels E, F) cells were mixed with approximately equal numbers of GFP-expressing BY4743 cells and inoculated into zinc-replete (LZM+100 µM ZnCl₂, panels A, C, E) or zinc-limiting (LZM+1 µM ZnCl₂, panels B, D, F) media and grown for fifteen generations prior to analysis by flow cytometry. Approximately 20,000 total cells per culture were assessed for GFP fluorescence (x-axis) and autofluorescence (y-axis). The <i>red line</i> in each panel marks the boundary between the sub-populations of tagged and untagged cells. Quantitation of these data is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002699#pgen-1002699-t001" target="_blank">Table 1</a>. The elongated distribution of fluorescence in zinc-limited cells is likely due to alterations in cell size and cell wall composition relative to zinc-replete cells and was observed for both GFP fluorescence and autofluorescence.</p