Yang Hui, Qu Liang, Ni Jianqiang, Wang Mengxi, Huang Youguo
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, PR China.
Mol Membr Biol. 2008 Jan;25(1):58-71. doi: 10.1080/09687680701528697.
Much in vivo and in vitro evidence has shown that the alpha subunits of heterotrimeric GTP-binding proteins (G proteins) exist as oligomers in their base state and disaggregate when being activated. In this article, the influence of palmitoylation modification of Galpha(o) on its oligomerization was explored extensively. Galpha(o) protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Galpha(o)) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Galpha(o) existed to a small extent as monomers but mostly as oligomers including dimers, trimers, tetramers and pentamers which could disaggregate completely into monomers by GTPgammaS stimulation. Palmitoylated Galpha(o), on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Galpha(o) was further investigated by several other biochemical and biophysical methods including gel filtration chromatography, analytical ultracentrifugation and atomic force microscopy analysis. The results consistently demonstrated that palmitoylation facilitated oligomerization of the Galpha(o) protein. Autoradiography indicated that [(14)C]-palmitoylated Galpha(o) would in no case disaggregate into monomers after treatment with GTPgammaS. [(35)S]-GTPgammaS binding activity assay showed that palmitoylated Galpha(o) was saturated at only 7.8 nmol/mg compared to 21.8 nmol/mg for non-palmitoylated Galpha(o). Fluorescent quenching studies using BODIPY FL-GTPgammaS as a probe showed that the conformation of GTP-binding domain of Galpha(o) tended to become more compact after palmitoylation. These results implied that palmitoylation may regulate the GDP/GTP exchange of Galpha(o) by influencing the oligomerization state of Galpha(o) and thereby modulate the on-off switch of the G protein in G protein-coupled signal transduction.
许多体内和体外证据表明，异源三聚体GTP结合蛋白（G蛋白）的α亚基在其基础状态下以寡聚体形式存在，并在激活时解聚。在本文中，广泛探讨了Gα(o)的棕榈酰化修饰对其寡聚化的影响。Gα(o)蛋白从与pQE60(Gα(o))和pBB131（N-肉豆蔻酰转移酶）共转化的大肠杆菌JM109菌株中表达和纯化。非变性凝胶电泳分析表明，Gα(o)在小程度上以单体形式存在，但大多以包括二聚体、三聚体、四聚体和五聚体的寡聚体形式存在，这些寡聚体可通过GTPγS刺激完全解聚为单体。另一方面，棕榈酰化的Gα(o)仅以难以解聚为单体的寡聚体形式存在。通过凝胶过滤色谱、分析超速离心和原子力显微镜分析等其他几种生化和生物物理方法，进一步研究了棕榈酰化对Gα(o)寡聚化的影响。结果一致表明，棕榈酰化促进了Gα(o)蛋白的寡聚化。放射自显影表明，[(14)C] - 棕榈酰化的Gα(o)在用GTPγS处理后绝不会解聚为单体。[(35)S] - GTPγS结合活性测定表明，棕榈酰化的Gα(o)的饱和浓度仅为7.8 nmol/mg，而非棕榈酰化的Gα(o)为21.8 nmol/mg。使用BODIPY FL - GTPγS作为探针的荧光猝灭研究表明，棕榈酰化后Gα(o)的GTP结合结构域的构象趋于变得更加紧凑。这些结果表明，棕榈酰化可能通过影响Gα(o)的寡聚化状态来调节Gα(o)的GDP/GTP交换，从而在G蛋白偶联信号转导中调节G蛋白的开关。
