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

A possible role for ICE2 in ER zinc homeostasis.A) Confirmation of the ice2Δ low zinc growth defect. Wild type (BY4743, filled columns) and ice2Δ (BY4743 ice2Δ, open columns) 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 ice2Δ mutant (BY4743 ice2Δ) 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 msc2Δ zrg17Δ zrc1Δ cot1Δ quadruple mutants. JSY5 (msc2Δ zrg17Δ zrc1Δ cot1Δ) and JSY5 ice2Δ (msc2Δ zrg17Δ zrc1Δ cot1Δ ice2Δ) 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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002699-g005: A possible role for ICE2 in ER zinc homeostasis.A) Confirmation of the ice2Δ low zinc growth defect. Wild type (BY4743, filled columns) and ice2Δ (BY4743 ice2Δ, open columns) 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 ice2Δ mutant (BY4743 ice2Δ) 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 msc2Δ zrg17Δ zrc1Δ cot1Δ quadruple mutants. JSY5 (msc2Δ zrg17Δ zrc1Δ cot1Δ) and JSY5 ice2Δ (msc2Δ zrg17Δ zrc1Δ cot1Δ ice2Δ) 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.

Mentions: As another illustration of the power of functional profiling to identify novel genes important for low zinc growth, we focused our attention on the ICE2 gene. ICE2 was previously identified as being needed for proper ER morphology [33]. Ice2 is an integral membrane protein with eight potential transmembrane domains suggesting that it is a transporter of some type. Our previous results indicated that the Msc2/Zrg17 zinc transporter complex plays the major role in maintaining ER zinc while the vacuolar Zrc1 and Cot1 zinc transporters also contribute [23], [34]. Our results had also indicated that additional ER zinc transporters were present and we therefore hypothesized that the substrate for Ice2-mediated transport was zinc. Consistent with that hypothesis, ice2Δ mutants grew poorly in low zinc (Table 3). We confirmed this growth defect by inoculating wild type and ice2Δ mutant cells into batch cultures and measuring culture density after overnight growth (Figure 5A). The ice2Δ mutants grew poorly relative to wild-type cells in LZM+1 µM Zn but adding as little as 3 µM Zn to the medium was sufficient to restore near wild-type growth. Higher concentrations were similarly effective (data not shown). This result indicated that Ice2 is required for optimal growth under severe zinc-limiting conditions but not under more moderate conditions. Zinc-deficient wild-type cells have induced expression of the ER-stress induced unfolded protein response (UPR) and this induction is exacerbated in mutants disrupted for MSC2 or ZRG17[23], [34]. Zinc supplements suppress UPR induction of both wild type and mutant cells indicating that luminal ER zinc is required for function of this compartment. Consistent with ice2Δ disrupting ER zinc homeostasis, we found that a UPRE-lacZ reporter was also hyper-induced in a zinc-limited ice2Δ mutant relative to wild-type cells and this hyper-induction was suppressed by supplementing zinc at 10 µM or above (Figure 5B, data not shown). We also deleted the ICE2 gene in a strain in which all four genes known to contribute to ER zinc were mutated (i.e. msc2Δ zrg17Δ zrc1Δ cot1Δ). Loss of Ice2 function in this quadruple mutant background caused still greater induction of the UPR when zinc was supplied at a concentration of 3 µM but the UPR was still suppressible with higher concentrations of zinc (Figure 5C).


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)

A possible role for ICE2 in ER zinc homeostasis.A) Confirmation of the ice2Δ low zinc growth defect. Wild type (BY4743, filled columns) and ice2Δ (BY4743 ice2Δ, open columns) 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 ice2Δ mutant (BY4743 ice2Δ) 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 msc2Δ zrg17Δ zrc1Δ cot1Δ quadruple mutants. JSY5 (msc2Δ zrg17Δ zrc1Δ cot1Δ) and JSY5 ice2Δ (msc2Δ zrg17Δ zrc1Δ cot1Δ ice2Δ) 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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1002699-g005: A possible role for ICE2 in ER zinc homeostasis.A) Confirmation of the ice2Δ low zinc growth defect. Wild type (BY4743, filled columns) and ice2Δ (BY4743 ice2Δ, open columns) 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 ice2Δ mutant (BY4743 ice2Δ) 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 msc2Δ zrg17Δ zrc1Δ cot1Δ quadruple mutants. JSY5 (msc2Δ zrg17Δ zrc1Δ cot1Δ) and JSY5 ice2Δ (msc2Δ zrg17Δ zrc1Δ cot1Δ ice2Δ) 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.
Mentions: As another illustration of the power of functional profiling to identify novel genes important for low zinc growth, we focused our attention on the ICE2 gene. ICE2 was previously identified as being needed for proper ER morphology [33]. Ice2 is an integral membrane protein with eight potential transmembrane domains suggesting that it is a transporter of some type. Our previous results indicated that the Msc2/Zrg17 zinc transporter complex plays the major role in maintaining ER zinc while the vacuolar Zrc1 and Cot1 zinc transporters also contribute [23], [34]. Our results had also indicated that additional ER zinc transporters were present and we therefore hypothesized that the substrate for Ice2-mediated transport was zinc. Consistent with that hypothesis, ice2Δ mutants grew poorly in low zinc (Table 3). We confirmed this growth defect by inoculating wild type and ice2Δ mutant cells into batch cultures and measuring culture density after overnight growth (Figure 5A). The ice2Δ mutants grew poorly relative to wild-type cells in LZM+1 µM Zn but adding as little as 3 µM Zn to the medium was sufficient to restore near wild-type growth. Higher concentrations were similarly effective (data not shown). This result indicated that Ice2 is required for optimal growth under severe zinc-limiting conditions but not under more moderate conditions. Zinc-deficient wild-type cells have induced expression of the ER-stress induced unfolded protein response (UPR) and this induction is exacerbated in mutants disrupted for MSC2 or ZRG17[23], [34]. Zinc supplements suppress UPR induction of both wild type and mutant cells indicating that luminal ER zinc is required for function of this compartment. Consistent with ice2Δ disrupting ER zinc homeostasis, we found that a UPRE-lacZ reporter was also hyper-induced in a zinc-limited ice2Δ mutant relative to wild-type cells and this hyper-induction was suppressed by supplementing zinc at 10 µM or above (Figure 5B, data not shown). We also deleted the ICE2 gene in a strain in which all four genes known to contribute to ER zinc were mutated (i.e. msc2Δ zrg17Δ zrc1Δ cot1Δ). Loss of Ice2 function in this quadruple mutant background caused still greater induction of the UPR when zinc was supplied at a concentration of 3 µM but the UPR was still suppressible with higher concentrations of zinc (Figure 5C).

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