title: Sst2 is a GTPase-activating protein for Gpa1: purification and characterization of a cognate RGS-Galpha protein pair in yeast.
abstract: Genetic studies in the yeast Saccharomyces cerevisiae have shown that SST2 promotes pheromone desensitization in vivo. Sst2 is the founding member of the RGS (regulators of G protein signaling) family of proteins, which in mammals act as GAPs (GTPase activating proteins) for several subfamilies of Galpha proteins in vitro. A similar activity for Sst2 has not been demonstrated, and it is not self-evident from sequence homology arguments alone. Here we describe the purification of Sst2 and its cognate Galpha protein (Gpa1) in yeast, and demonstrate Sst2-stimulated Gpa1 GTPase activity. His-tagged versions of Sst2 and Gpa1 were expressed in E. coli, and purified using Ni2+-agarose and ion exchange chromatography. Time-course binding experiments reveal that Sst2 does not affect the binding or release of guanine nucleotides. Similarly, steady-state GTPase assays reveal that Sst2 does not alter the overall rate of hydrolysis, including the rate-limiting nucleotide exchange step. Single-turnover GTPase assays reveal, however, that Sst2 is a potent stimulator of GTP hydrolysis. Sst2 also exhibits GAP activity for mammalian Goalpha, and the mammalian RGS protein GAIP exhibits GAP activity for Gpa1. Finally, we show that Sst2 binds with highest affinity to the transition state of Gpa1 (GDP-AlF4--bound), and with much lower affinity to the inactive (GDP-bound) and active (GTPgammaS-bound) conformations. These experiments represent the first biochemical characterization of Gpa1 and Sst2, and provide a molecular basis for their well-established biological roles in signaling and desensitization.
authors.affiliation: Department of Pharmacology and Molecular Cardiobiology Program, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Apanovitch
authors.forename: K C
authors.initials: KC
authors.lastname: Slep
authors.forename: P B
authors.initials: PB
authors.lastname: Sigler
authors.forename: H G
authors.initials: HG
authors.lastname: Dohlman
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins; D006868:Hydrolysis; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D020710:RGS Proteins
chemical_list: D005656:Fungal Proteins; D020558:GTP Phosphohydrolases; D044385:GTP-Binding Protein alpha Subunits; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D020962:Heterotrimeric GTP-Binding Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; C094301:SST2 protein, S cerevisiae; D020710:RGS Proteins; C084293:GPA1 protein, S cerevisiae