A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae.
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.
Department of Bioengineering, University of California San Diego, La Jolla, CA 92093-0412, USA.
Chris
C
Grilly
Jesse
J
Stricker
Wyming Lee
WL
Pang
Matthew R
MR
Bennett
Jeff
J
Hasty
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000378:metabolism; D049071:Endopeptidase Clp / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D053263:Gene Regulatory Networks; D049452:Green Fluorescent Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities