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A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae.

Grilly C, Stricker J, Pang WL, Bennett MR, Hasty J - Mol. Syst. Biol. (2007)

Bottom Line: Using a microfluidic platform tailored for single-cell fluorescence measurements, we monitor protein decay rates after repression using an ssrA-tagged fluorescent reporter.We observe a half-life ranging from 91 to 22 min, depending on the level of activation of the degradation genes.Computational modeling of the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations, implying that degradation is governed by Michaelis-Menten-type interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California San Diego, La Jolla, CA 92093-0412, USA.

ABSTRACT
Protein decay rates are regulated by degradation machinery that clears unnecessary housekeeping proteins and maintains appropriate dynamic resolution for transcriptional regulators. Turnover rates are also crucial for fluorescence reporters that must strike a balance between sufficient fluorescence for signal detection and temporal resolution for tracking dynamic responses. Here, we use components of the Escherichia coli degradation machinery to construct a Saccharomyces cerevisiae strain that allows for tunable degradation of a tagged protein. Using a microfluidic platform tailored for single-cell fluorescence measurements, we monitor protein decay rates after repression using an ssrA-tagged fluorescent reporter. We observe a half-life ranging from 91 to 22 min, depending on the level of activation of the degradation genes. Computational modeling of the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations, implying that degradation is governed by Michaelis-Menten-type interactions. In addition to providing a reporter with tunable dynamic resolution, our findings set the stage for explorations of the effect of protein degradation on gene regulatory and signalling pathways.

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Effect of the exogenous degradation machinery on CGD699. (A) Doubling times in batch culture for various strains and IPTG concentrations. Shown are the original strain (K699), two intermediate strains (K699 with yEGFP and K699 with yEGFP and ClpXP) and the complete strain (CGD699). (B) Flow cytometry forward scatter means for K699 and CGD699 for various IPTG concentrations. Note that varying the IPTG level (and therefore the resulting concentration of ClpXP) does not significantly affect the forward scatter. (C) Flow cytometry fluorescence means for CGD759 derivatives containing either untagged or ssrA-tagged yEGFP integration cassettes. CGD759 contains integrated clpP, clpX and lacI expression cassettes as in CGD699. Note that the addition of IPTG causes a loss of fluorescence only with ssrA-tagged yEGFP.
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f2: Effect of the exogenous degradation machinery on CGD699. (A) Doubling times in batch culture for various strains and IPTG concentrations. Shown are the original strain (K699), two intermediate strains (K699 with yEGFP and K699 with yEGFP and ClpXP) and the complete strain (CGD699). (B) Flow cytometry forward scatter means for K699 and CGD699 for various IPTG concentrations. Note that varying the IPTG level (and therefore the resulting concentration of ClpXP) does not significantly affect the forward scatter. (C) Flow cytometry fluorescence means for CGD759 derivatives containing either untagged or ssrA-tagged yEGFP integration cassettes. CGD759 contains integrated clpP, clpX and lacI expression cassettes as in CGD699. Note that the addition of IPTG causes a loss of fluorescence only with ssrA-tagged yEGFP.

Mentions: We have constructed a S. cerevisiae strain (CGD699) that allows tunable degradation of a tagged protein. To accomplish this, we expressed a modified E. coli ClpXP protease in yeast under the control of a repressible promoter. Proteins that are tagged with the ssrA tag are quickly degraded, and this degradation rate is controlled by the induction level of ClpXP. We integrated the two E. coli genes (clpX and clpP) that code for the ClpXP protease into the yeast genome (Figure 1, and Materials and methods). We found that the clpX gene needed to be modified with 10 silent mutations to be expressed in yeast (see Materials and methods). These two genes were placed under the control of two separate copies of the modified ADH1i promoter (Blake, 2003), at two different loci in the yeast genome. Additionally, we integrated mlacI, a mammalian-enhanced version of lacI (Cronin et al, 2001), controlled by a wild-type ADH1 promoter. The wild-type version of the ADH1 promoter exhibits constitutive expression, while the ADH1i promoter is repressed by LacI in the absence of IPTG. Addition of IPTG to the medium results in ClpXP production and degradation of a tagged protein. To demonstrate the utility of this approach, we also integrated a yEGFP gene tagged with an 11-amino-acid ssrA tag (AANDENYALAA), under the control of the GAL1 promoter into CGD699. This promoter is fully induced by 0.5% w/v galactose and repressed by 2% w/v glucose. CGD699 cells grown in the presence of galactose produce GFP and are fluorescent, and GFP production ceases if the carbon source in the media is switched from galactose to glucose. Observations of growth rate and morphology indicate that the exogenous proteases cause no deleterious cellular effects over a wide range of IPTG levels (Figure 2A and B). While coexpression of tagged yEGFP and the degradation machinery resulted in almost complete loss of fluorescence, coexpression of untagged yEGFP with the degradation machinery showed no significant drop in fluorescence (Figure 2C). This confirms that the degradation effect is specific to tagged proteins.


A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae.

Grilly C, Stricker J, Pang WL, Bennett MR, Hasty J - Mol. Syst. Biol. (2007)

Effect of the exogenous degradation machinery on CGD699. (A) Doubling times in batch culture for various strains and IPTG concentrations. Shown are the original strain (K699), two intermediate strains (K699 with yEGFP and K699 with yEGFP and ClpXP) and the complete strain (CGD699). (B) Flow cytometry forward scatter means for K699 and CGD699 for various IPTG concentrations. Note that varying the IPTG level (and therefore the resulting concentration of ClpXP) does not significantly affect the forward scatter. (C) Flow cytometry fluorescence means for CGD759 derivatives containing either untagged or ssrA-tagged yEGFP integration cassettes. CGD759 contains integrated clpP, clpX and lacI expression cassettes as in CGD699. Note that the addition of IPTG causes a loss of fluorescence only with ssrA-tagged yEGFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1943424&req=5

f2: Effect of the exogenous degradation machinery on CGD699. (A) Doubling times in batch culture for various strains and IPTG concentrations. Shown are the original strain (K699), two intermediate strains (K699 with yEGFP and K699 with yEGFP and ClpXP) and the complete strain (CGD699). (B) Flow cytometry forward scatter means for K699 and CGD699 for various IPTG concentrations. Note that varying the IPTG level (and therefore the resulting concentration of ClpXP) does not significantly affect the forward scatter. (C) Flow cytometry fluorescence means for CGD759 derivatives containing either untagged or ssrA-tagged yEGFP integration cassettes. CGD759 contains integrated clpP, clpX and lacI expression cassettes as in CGD699. Note that the addition of IPTG causes a loss of fluorescence only with ssrA-tagged yEGFP.
Mentions: We have constructed a S. cerevisiae strain (CGD699) that allows tunable degradation of a tagged protein. To accomplish this, we expressed a modified E. coli ClpXP protease in yeast under the control of a repressible promoter. Proteins that are tagged with the ssrA tag are quickly degraded, and this degradation rate is controlled by the induction level of ClpXP. We integrated the two E. coli genes (clpX and clpP) that code for the ClpXP protease into the yeast genome (Figure 1, and Materials and methods). We found that the clpX gene needed to be modified with 10 silent mutations to be expressed in yeast (see Materials and methods). These two genes were placed under the control of two separate copies of the modified ADH1i promoter (Blake, 2003), at two different loci in the yeast genome. Additionally, we integrated mlacI, a mammalian-enhanced version of lacI (Cronin et al, 2001), controlled by a wild-type ADH1 promoter. The wild-type version of the ADH1 promoter exhibits constitutive expression, while the ADH1i promoter is repressed by LacI in the absence of IPTG. Addition of IPTG to the medium results in ClpXP production and degradation of a tagged protein. To demonstrate the utility of this approach, we also integrated a yEGFP gene tagged with an 11-amino-acid ssrA tag (AANDENYALAA), under the control of the GAL1 promoter into CGD699. This promoter is fully induced by 0.5% w/v galactose and repressed by 2% w/v glucose. CGD699 cells grown in the presence of galactose produce GFP and are fluorescent, and GFP production ceases if the carbon source in the media is switched from galactose to glucose. Observations of growth rate and morphology indicate that the exogenous proteases cause no deleterious cellular effects over a wide range of IPTG levels (Figure 2A and B). While coexpression of tagged yEGFP and the degradation machinery resulted in almost complete loss of fluorescence, coexpression of untagged yEGFP with the degradation machinery showed no significant drop in fluorescence (Figure 2C). This confirms that the degradation effect is specific to tagged proteins.

Bottom Line: Using a microfluidic platform tailored for single-cell fluorescence measurements, we monitor protein decay rates after repression using an ssrA-tagged fluorescent reporter.We observe a half-life ranging from 91 to 22 min, depending on the level of activation of the degradation genes.Computational modeling of the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations, implying that degradation is governed by Michaelis-Menten-type interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California San Diego, La Jolla, CA 92093-0412, USA.

ABSTRACT
Protein decay rates are regulated by degradation machinery that clears unnecessary housekeeping proteins and maintains appropriate dynamic resolution for transcriptional regulators. Turnover rates are also crucial for fluorescence reporters that must strike a balance between sufficient fluorescence for signal detection and temporal resolution for tracking dynamic responses. Here, we use components of the Escherichia coli degradation machinery to construct a Saccharomyces cerevisiae strain that allows for tunable degradation of a tagged protein. Using a microfluidic platform tailored for single-cell fluorescence measurements, we monitor protein decay rates after repression using an ssrA-tagged fluorescent reporter. We observe a half-life ranging from 91 to 22 min, depending on the level of activation of the degradation genes. Computational modeling of the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations, implying that degradation is governed by Michaelis-Menten-type interactions. In addition to providing a reporter with tunable dynamic resolution, our findings set the stage for explorations of the effect of protein degradation on gene regulatory and signalling pathways.

Show MeSH
Related in: MedlinePlus