Limits...
Genome-wide functional profiling identifies genes and processes important for zinc-limited growth of Saccharomyces cerevisiae.

North M, Steffen J, Loguinov AV, Zimmerman GR, Vulpe CD, Eide DJ - PLoS Genet. (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Science and Toxicology, University of California Berkeley, Berkeley, California, USA.

ABSTRACT
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.

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Analysis of low zinc growth by flow cytometry.Untagged wild-type BY4743 (panels A, B), gal2Δ (panels C, D), and tsa1Δ (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 red line in each panel marks the boundary between the sub-populations of tagged and untagged cells. Quantitation of these data is provided in Table 1. 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.
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pgen-1002699-g002: Analysis of low zinc growth by flow cytometry.Untagged wild-type BY4743 (panels A, B), gal2Δ (panels C, D), and tsa1Δ (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 red line in each panel marks the boundary between the sub-populations of tagged and untagged cells. Quantitation of these data is provided in Table 1. 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.

Mentions: To confirm these results for selected mutants, we used an independent and sensitive assay of growth using flow cytometry. The wild-type strain (BY4743) was transformed with an integrating plasmid vector that expressed GFP from a strong promoter [26]. GFP-expressing cells can be readily distinguished from untagged cells by flow cytometry. To compare growth of a GFP-tagged wild-type strain with an untagged mutant, the two strains were mixed together with approximately equal numbers of cells and then inoculated into low zinc or zinc-replete media. After fifteen generations, the level of each strain was assessed by counting ∼20,000 total cells per culture. As shown in Figure 2A and 2B, GFP-tagged and untagged wild-type cells grew equally well in both low zinc and zinc-replete conditions. Quantitation of the results from triplicate cultures is provided in Table 1. As an additional control, we tested growth of an untagged gal2Δ mutant. GAL2 encodes galactose permease and is not predicted to be required for growth in the medium used here where glucose is the carbon source. No growth defect was observed (Figure 2C and D, Table 1) indicating that the antibiotic resistance marker (kanMX4) used to generate the deletion mutants in the collection does not alter growth in low zinc or zinc-replete media. In contrast, while the untagged tsa1Δ mutant grew fairly well in the zinc-replete medium and was found at ∼28% of the final population after 15 generations, it was almost completely out-competed by the wild-type strain in low zinc cultures (0.6%, Figure 2E versus Figure 2F, Table 1).


Genome-wide functional profiling identifies genes and processes important for zinc-limited growth of Saccharomyces cerevisiae.

North M, Steffen J, Loguinov AV, Zimmerman GR, Vulpe CD, Eide DJ - PLoS Genet. (2012)

Analysis of low zinc growth by flow cytometry.Untagged wild-type BY4743 (panels A, B), gal2Δ (panels C, D), and tsa1Δ (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 red line in each panel marks the boundary between the sub-populations of tagged and untagged cells. Quantitation of these data is provided in Table 1. 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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3369956&req=5

pgen-1002699-g002: Analysis of low zinc growth by flow cytometry.Untagged wild-type BY4743 (panels A, B), gal2Δ (panels C, D), and tsa1Δ (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 red line in each panel marks the boundary between the sub-populations of tagged and untagged cells. Quantitation of these data is provided in Table 1. 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.
Mentions: To confirm these results for selected mutants, we used an independent and sensitive assay of growth using flow cytometry. The wild-type strain (BY4743) was transformed with an integrating plasmid vector that expressed GFP from a strong promoter [26]. GFP-expressing cells can be readily distinguished from untagged cells by flow cytometry. To compare growth of a GFP-tagged wild-type strain with an untagged mutant, the two strains were mixed together with approximately equal numbers of cells and then inoculated into low zinc or zinc-replete media. After fifteen generations, the level of each strain was assessed by counting ∼20,000 total cells per culture. As shown in Figure 2A and 2B, GFP-tagged and untagged wild-type cells grew equally well in both low zinc and zinc-replete conditions. Quantitation of the results from triplicate cultures is provided in Table 1. As an additional control, we tested growth of an untagged gal2Δ mutant. GAL2 encodes galactose permease and is not predicted to be required for growth in the medium used here where glucose is the carbon source. No growth defect was observed (Figure 2C and D, Table 1) indicating that the antibiotic resistance marker (kanMX4) used to generate the deletion mutants in the collection does not alter growth in low zinc or zinc-replete media. In contrast, while the untagged tsa1Δ mutant grew fairly well in the zinc-replete medium and was found at ∼28% of the final population after 15 generations, it was almost completely out-competed by the wild-type strain in low zinc cultures (0.6%, Figure 2E versus Figure 2F, Table 1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Nutritional Science and Toxicology, University of California Berkeley, Berkeley, California, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus