A quantitative characterization of the yeast heterotrimeric G protein cycle

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)-Gα and Gβγ-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 α-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.

[1]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[2]  R. K. Chan,et al.  Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones , 1982, Molecular and cellular biology.

[3]  M. Leider.  Goodman & Gilman's The Pharmacological Basis of Therapeutics , 1985 .

[4]  W. Stigelman,et al.  Goodman and Gilman's the Pharmacological Basis of Therapeutics , 1986 .

[5]  L. Hartwell,et al.  The C-terminus of the S. cerevisiae α-pheromone receptor mediates an adaptive response to pheromone , 1988, Cell.

[6]  J. Thorner,et al.  The carboxy-terminal segment of the yeast α-factor receptor is a regulatory domain , 1988, Cell.

[7]  Leland H. Hartwell,et al.  Courtship in S. cerevisiae: Both cell types choose mating partners by responding to the strongest pheromone signal , 1990, Cell.

[8]  Janina Maier,et al.  Guide to yeast genetics and molecular biology. , 1991, Methods in enzymology.

[9]  T. Lamb,et al.  Amplification and kinetics of the activation steps in phototransduction. , 1993, Biochimica et biophysica acta.

[10]  AC Tose.  Cell , 1993, Cell.

[11]  J. Rohrer,et al.  Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast. , 1993, Molecular biology of the cell.

[12]  Gerald R. Fink,et al.  Guide to yeast genetics and molecular biology , 1993 .

[13]  Henry R. Bourne,et al.  Lipid Modifications of Trimeric G Proteins (*) , 1995, The Journal of Biological Chemistry.

[14]  H. Dohlman,et al.  Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[15]  Howard Riezman,et al.  Ubiquitination of a Yeast Plasma Membrane Receptor Signals Its Ligand-Stimulated Endocytosis , 1996, Cell.

[16]  J. Thorner,et al.  Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit) , 1996, Molecular and cellular biology.

[17]  Solomon H. Snyder,et al.  Drugs and the brain , 1996 .

[18]  H. Hamm,et al.  The 2.0 Å crystal structure of a heterotrimeric G protein , 1996, Nature.

[19]  K. Blumer,et al.  RGS family members: GTPase-activating proteins for heterotrimeric G-protein α-subunits , 1996, Nature.

[20]  H. Dohlman,et al.  Regulation of Membrane and Subunit Interactions by N-Myristoylation of a G Protein α Subunit in Yeast* , 1996, The Journal of Biological Chemistry.

[21]  C. Cowan,et al.  RGS9, a GTPase Accelerator for Phototransduction , 1998, Neuron.

[22]  P B Sigler,et al.  Sst2 is a GTPase-activating protein for Gpa1: purification and characterization of a cognate RGS-Galpha protein pair in yeast. , 1998, Biochemistry.

[23]  J. Linderman,et al.  A mathematical model for ligand/receptor/G-protein dynamics and actin polymerization in human neutrophils. , 1998, Journal of theoretical biology.

[24]  J. Hepler.  Emerging roles for RGS proteins in cell signalling. , 1999, Trends in pharmacological sciences.

[25]  T. Lamb,et al.  The Gain of Rod Phototransduction Reconciliation of Biochemical and Electrophysiological Measurements , 2000, Neuron.

[26]  Wei He,et al.  Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1 , 2000, Nature.

[27]  S. Ferguson,et al.  Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. , 2001, Pharmacological reviews.

[28]  P. Devreotes,et al.  Receptor-Mediated Activation of Heterotrimeric G-Proteins in Living Cells , 2001, Science.

[29]  D. Wilkin,et al.  Neuron , 2001, Brain Research.

[30]  J. Thorner,et al.  Regulation of G protein-initiated signal transduction in yeast: paradigms and principles. , 2001, Annual review of biochemistry.