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.

Show MeSH

Related in: MedlinePlus

Functional profiling analysis.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 tsa1Δ 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3369956&req=5

pgen-1002699-g001: Functional profiling analysis.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 tsa1Δ 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.

Mentions: To identify genes important for low zinc growth, cells were grown in either zinc-limiting (LZM+1 µM ZnCl2) or zinc-replete (LZM+100 µM ZnCl2) media. LZM allows for control of the zinc available to cells due to the high concentrations of EDTA (1 mM) and citrate (20 mM) serving as a metal buffers in that medium. Wild-type cells grew markedly better in LZM+100 µM ZnCl2 [area under curve (AUC) value = 34.7] than did cells grown in LZM+1 µM ZnCl2 (AUC = 21.0) (Figure 1A). Thus, cells grown in LZM+1 µM ZnCl2 are zinc limited. Supplementation of 100 µM Zn was chosen as the replete condition because it provides sufficient zinc for maximal growth but does not exceed the metal buffering capacity of the medium and alter the availability of other metals such as Fe and Cu [18]. As an illustration of defective growth in low zinc, we included a tsa1Δ mutant in this experiment. TSA1 encodes the major cytosolic peroxiredoxin in yeast and is required for combating the oxidative stress encountered during low zinc growth [22]. As shown in Figure 1A, the tsa1Δ mutant grew almost as well as the wild-type strain in LZM+100 µM ZnCl2 but very poorly in LZM+1 µM ZnCl2. Our previous studies demonstrated that adding other metals to 100 µM concentrations did not increase growth rate in LZM+1 µM Zn of either wild type or tsa1Δ mutants indicating that these cells are specifically limited for zinc [22].


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)

Functional profiling analysis.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 tsa1Δ 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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002699-g001: Functional profiling analysis.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 tsa1Δ 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.
Mentions: To identify genes important for low zinc growth, cells were grown in either zinc-limiting (LZM+1 µM ZnCl2) or zinc-replete (LZM+100 µM ZnCl2) media. LZM allows for control of the zinc available to cells due to the high concentrations of EDTA (1 mM) and citrate (20 mM) serving as a metal buffers in that medium. Wild-type cells grew markedly better in LZM+100 µM ZnCl2 [area under curve (AUC) value = 34.7] than did cells grown in LZM+1 µM ZnCl2 (AUC = 21.0) (Figure 1A). Thus, cells grown in LZM+1 µM ZnCl2 are zinc limited. Supplementation of 100 µM Zn was chosen as the replete condition because it provides sufficient zinc for maximal growth but does not exceed the metal buffering capacity of the medium and alter the availability of other metals such as Fe and Cu [18]. As an illustration of defective growth in low zinc, we included a tsa1Δ mutant in this experiment. TSA1 encodes the major cytosolic peroxiredoxin in yeast and is required for combating the oxidative stress encountered during low zinc growth [22]. As shown in Figure 1A, the tsa1Δ mutant grew almost as well as the wild-type strain in LZM+100 µM ZnCl2 but very poorly in LZM+1 µM ZnCl2. Our previous studies demonstrated that adding other metals to 100 µM concentrations did not increase growth rate in LZM+1 µM Zn of either wild type or tsa1Δ mutants indicating that these cells are specifically limited for zinc [22].

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