Fig. 4.
Quantitative protein turnover measurements during heat shock reveal UBI4 repeat number-dependent degradation kinetics. a Experimental design for turnover measurements using fluorescent protein reporters. UBI4 repeat variants expressing UbG76V-GFP (a substrate for UPS-mediated proteolysis) or Ub-M-GFP (stable GFP)27, 28 were used to measure the UPS activity during heat shock (44 °C). Cells were grown at 30 °C until exponential phase in SC medium with 2% galactose to induce the expression of the fluorescent reporters. The cultures were then shifted to 44 °C and samples were taken before the temperature shift (T0) and at multiple time points after for flow cytometry analysis. For each time point, the fraction of fluorescent cells over the total cell population was calculated. b We then fit the fraction to a first-order decay function to calculate the degradation rate constant K deg. Since growth is completely arrested during the heat shock period, we do not expect any contribution from protein dilution rates (as a result of cell division). Protein half-lives were calculated using the formula T (1/2) = ln (2)/K deg. c The initial proportion (i.e., before heat shock) of fluorescent cells over the total population gives the steady-state levels of the reporter proteins and reflects the UPS activity under physiological conditions. b, c Data are mean of 4 independent replicates ± SEM