SST2 IS A GTPASE-ACTIVATING PROTEIN FOR GPA1 - PURIFICATION AND CHARACTERIZATION OF A COGNATE RGS-G-ALPHA PROTEIN PAIR IN YEAST

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 protei ns, which in mammals act as GAPs (GTPase activating proteins) for seve ral subfamilies of G alpha proteins in vitro. A similar activity for S st2 has not been demonstrated, and it is not self-evident from sequenc e homology arguments alone. Here we describe the purification of Sst2 and its cognate Get protein (Gpa1) in yeast, and demonstrate Sst2-stim ulated Gpa1 GTPase activity. His-tagged versions of Sst2 and Gpa1 were expressed in E. coli, and purified using Ni2+-agarose and ion exchang e chromatography. Time-course binding experiments reveal that Sst2 doe s not affect the binding or release of guanine nucleotides. Similarly, steady-state GTPase assays reveal that Sst2 does not alter the overal l rate of hydrolysis, including the rate-limiting nucleotide exchange step. Single-turnover GTPase assays reveal, however, that Sst2 is a po tent stimulator of GTP hydrolysis. Sst2 also exhibits GAP activity for mammalian G(o) alpha, and the mammalian RGS protein GAIP exhibits GAP activity for Gpa1. Finally, we show that Sst2 binds with highest affi nity to the transition state of Gpa1 (GDP-AlF4--bound), and with much lower affinity to the inactive (GDP-bound) and active (GTP gamma S-bou nd) conformations. These experiments represent the first biochemical c haracterization of Gpa1 and Sst2, and provide a molecular basis for th eir well-established biological roles in signaling and desensitization .