A quantitative characterization of the yeast heterotrimeric G protein cycle

Abstract

The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Galpha and Gbetagamma-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose-response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of alpha-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose-response curve and the downstream dose-response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast.

Fig. 1.

Reaction diagram of heterotrimeric G protein cycle. The individual reactions comprising the key dynamics of heterotrimeric G proteins in yeast are represented along with the rate constants. The solid dot on the reaction arrow indicates that the reaction is catalyzed by the protein connected to the dot. The yellow circle attached to Gβγ represents YFP fused to Ste18p (Gγ) and the blue circle attached to Gα represents CFP fused to Gpa1p (Gα).

Fig. 2.

G protein activation results in a loss of FRET. Shown is a representative fluorescence spectra of TMY101 showing FRET. Cells were excited at 430 nm, and the emission was detected by scanning from 466 to 600 nm. The control (dashed-dot line) was the combined signal from TMY102 (CFP-Gpa1p) and TMY103 (Ste18p-YFP). FRET was detected in TMY101 (solid line) as a decrease in the CFP emission signal (475 nm) and an increase in the YFP emission spectra (530 nm) relative to the control. Treating the TMY101 cells with α-factor for 1 min resulted in a loss of FRET (dashed line).

Fig. 3.

Kinetics of G protein activation. (A) Time course of G protein activation. TMY101 cells were treated with 1 μM α-factor, and the FRET emission ratio 475/530 was measured at different time points from 0 to 10 min (black). These values were normalized by subtracting the baseline ratio at t = 0, and then dividing by the peak baseline adjusted ratio at t = 30 s. In a second experiment, the protein synthesis inhibitor cycloheximide was added with α-factor at the start (red). The time course of G protein activation at later times up to 60 min is also shown (Inset). The SE from three independent measurements are represented. (B) Time course of G protein activation in sst2Δ and ste2^300Δ cells. TMY111 (sst2Δ, blue) and TMY112 (ste2^300Δ, red) cells were treated with 1 μM α-factor, the FRET 475/530 ratio was measured from 0 to 10 min, and the baseline was adjusted and then normalized to the ratio at the 30-s time point. The data from TMY101 are redrawn in black for comparison.

Fig. 4.

Overlap of different dose–response curves in G protein signaling. (A) Pheromone dose–response behavior for TMY101 cells. α-Factor was added at different doses from 0.1 nM to 1 μM. Four different pheromone dose–response readouts were measured: (i) receptor affinity (black triangles), (ii) G protein activation (red circles), (iii) transcriptional induction of P_FUS1-GFP (green squares), and (iv) cell-cycle arrest (blue diamonds). The data points from each readout were normalized to the response at 1 μM and were then fit with a Hill curve. (B) Pheromone dose–response behavior of TMY111 (sst2Δ) cells. α-Factor was added at different doses from 0.03 nM to 1 μM. The data from the four different dose–response readouts are presented as described above.

Fig. 5.

Fitting the mathematical model to the TMY101 data. (A) Experimental data and model simulations describing the time course of G protein activation in TMY101. Two of the model parameters (k_Ga and k_Gd1) were fit to the data. The normalized 475/530 data were processed into the fraction of total G proteins that are active (•). A simulation of the fitted model in response to an input of 1 μM α-factor is presented (black line). (B) Experiments and modeling describing the G protein activation dose–response behavior of TMY101. The data (•) and simulations (black line) were normalized to the output value at 1 μM α-factor. A time point of t = 60 s was used. The error bars represent the SE from three independent measurements.