Landry S, Hoffman C S
Department of Biology, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Genetics. 2001 Mar;157(3):1159-68. doi: 10.1093/genetics/157.3.1159.
Fission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein. The gpa2 Galpha and git5 Gbeta are both required for glucose-triggered cAMP signaling. The git5 Gbeta is a unique member of the Gbeta family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gbeta folding and interaction with Ggamma subunits. Using a git5 bait in a two-hybrid screen, we identified the git11 Ggamma gene. Co-immunoprecipitation studies confirm the composition of this Gbetagamma dimer. Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Galpha or the git5 Gbeta. Overexpression of the gpa2 Galpha partially suppresses loss of either the git5 Gbeta or the git11 Ggamma, while mutational activation of the Galpha fully suppresses loss of either Gbeta or Ggamma. Deletion of gpa2 (Galpha), git5 (Gbeta), or git11 (Ggamma) confer quantitatively distinct effects on fbp1 repression, indicating that the gpa2 Galpha subunit remains partially active in the absence of the Gbetagamma dimer and that the git5 Gbeta subunit remains partially active in the absence of the git11 Ggamma subunit. The addition of the CAAX box from the git11 Ggamma to the carboxy-terminus of the git5 Gbeta partially suppresses the loss of the Ggamma. Thus the Ggamma in this system is presumably required for localization of the Gbetagamma dimer but not for folding of the Gbeta subunit. In mammalian cells, the essential roles of the Gbeta amino-terminal coiled-coil domains and Ggamma partners in Gbeta folding may therefore reflect a mechanism used by cells that express multiple forms of both Gbeta and Ggamma subunits to regulate the composition and activity of its G proteins.
裂殖酵母腺苷酸环化酶与哺乳动物腺苷酸环化酶一样,受异源三聚体G蛋白调控。gpa2 Gα和git5 Gβ对于葡萄糖触发的cAMP信号传导都是必需的。git5 Gβ是Gβ家族的一个独特成员,因为它缺乏一个氨基末端卷曲螺旋结构域,而该结构域对于哺乳动物Gβ折叠以及与Gγ亚基相互作用至关重要。在双杂交筛选中使用git5诱饵,我们鉴定出了git11 Gγ基因。免疫共沉淀研究证实了这种Gβγ二聚体的组成。缺失git11的细胞在fbp1转录的葡萄糖抑制和有性发育方面存在缺陷,类似于缺乏gpa2 Gα或git5 Gβ的细胞。gpa2 Gα的过表达部分抑制了git5 Gβ或git11 Gγ的缺失,而Gα的突变激活则完全抑制了Gβ或Gγ的缺失。缺失gpa2(Gα)、git5(Gβ)或git11(Gγ)对fbp1抑制产生数量上不同的影响,这表明gpa2 Gα亚基在没有Gβγ二聚体时仍部分具有活性,并且git5 Gβ亚基在没有git11 Gγ亚基时仍部分具有活性。将git11 Gγ的CAAX框添加到git5 Gβ的羧基末端可部分抑制Gγ的缺失。因此,该系统中的Gγ可能是Gβγ二聚体定位所必需的,但不是Gβ亚基折叠所必需的。在哺乳动物细胞中,Gβ氨基末端卷曲螺旋结构域和Gγ伴侣在Gβ折叠中的重要作用可能反映了一种机制,即表达多种形式的Gβ和Gγ亚基的细胞利用该机制来调节其G蛋白的组成和活性。