Sst2 is a GTPase-activating protein for Gpa1:: Purification and characterization of a cognate RGS-Gα protein pair in yeast
Sst2 is a GTPase-activating protein for Gpa1:: Purification and characterization of a cognate RGS-Gα protein pair in yeast
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DOI：
10.1021/bi9729965
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发表时间：
1998-04-07
期刊：
影响因子：
2.9
通讯作者：
Dohlman, HG
中科院分区：
文献类型：
--
作者：
Apanovitch, DM;Slep, KC;Dohlman, HG
对酿酒酵母的遗传学研究表明，Sst2在体内促进信息素脱敏。Sst2是G蛋白信号调节蛋白(RGS)家族的创始成员，在哺乳动物中，RGS家族在体外作为Gα蛋白几个亚家族的GTP酶激活蛋白。Sst2的类似活性还没有被证明，单从序列同源性的争论来看也不是不言而喻的。在这里，我们描述了酵母中Sst2及其同源Get蛋白(Gpa1)的纯化，并证明了Sst2刺激Gpa1 GTP酶的活性。His标记的Sst2和Gpa1在大肠杆菌中表达，并经Ni2+-琼脂糖凝胶和离子交换层析纯化。时程结合实验表明，Sst2不影响鸟嘌呤核苷酸的结合或释放。同样，稳态GTP酶分析显示，Sst2不改变总的水解率，包括限速核苷酸交换步骤。然而，单周转GTP酶分析表明，Sst2是GTP水解酶的有效刺激因子。Sst2对哺乳动物G(O)α也有GAP活性，哺乳动物RGS蛋白GAIP对Gpa1也有GAP活性。最后，我们发现Sst2与Gpa1过渡态(Gdp-AlF4结合)的亲和力最高，而与非活动构象(Gdp结合)和活动构象(GTP Gamma S结合)的亲和力要低得多。这些实验代表了Gpa1和Sst2的第一个生化特征，并为它们在信号转导和脱敏中的生物学作用提供了分子基础。
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 G alpha 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 Get 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 G(o) alpha, 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 (GTP gamma S-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.