Matsuda T, Takao T, Shimonishi Y, Murata M, Asano T, Yoshizawa T, Fukada Y
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Japan.
J Biol Chem. 1994 Dec 2;269(48):30358-63.
The gamma-subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are isoprenylated and alpha-carboxyl methylated at their COOH-terminal cysteine residues. These modifications are necessary for membrane attachment of the beta gamma complex, but a requirement of an additional factor has been proposed for the stable binding. We explored a possible contribution of the blocked amino terminus of beta-subunits of bovine photoreceptor G-protein, transducin (T alpha/T beta gamma = Gt alpha/beta 1 gamma 1), and of three beta gamma complexes (beta 1 gamma 2, beta 1 gamma 3, and beta 1 gamma 7) purified from bovine brains. Structural analyses revealed that every beta 1-subunit has an N-acetylated serine, which is unlikely to contribute to the membrane association. Since neither protease nor heat treatment of photoreceptor membranes affected the membrane binding of T beta gamma, it seems unlikely that rhodopsin (or other membrane proteins) serves as an anchor protein for accepting T beta gamma. In fact, T beta gamma bound to phospholipid large unilamellar vesicles (LUVs), of which the polar head groups strongly influenced the binding: T beta gamma alone showed 2-fold higher binding for negatively charged phosphatidylserine-LUVs than for neutral phosphatidylcholine (PC)-LUVs, while the affinity of T alpha/T beta gamma complex for the phosphatidylserine-LUVs was lower than that for the PC-LUVs. These results indicate that 1) an ionic interaction between T beta gamma and membrane surface plays an important role in the stable membrane association, and 2) the domain(s) of T beta gamma responsible for the association would be different between trimeric and dissociated states. We also found that synthetic peptides corresponding to the COOH-terminal region of T gamma inhibited T alpha-T beta gamma interaction only when the peptides were isoprenylated. This suggests that the isoprenyl moiety is located at the contact site between the subunits, not at the membrane-binding domain, when T beta gamma is complexed with T alpha.
异三聚体鸟嘌呤核苷酸结合调节蛋白（G蛋白）的γ亚基在其COOH末端的半胱氨酸残基处进行异戊二烯化和α-羧甲基化修饰。这些修饰对于βγ复合物附着于膜是必需的，但有人提出稳定结合还需要另外一个因子。我们研究了牛感光G蛋白转导素（Tα/Tβγ = Gtα/β1γ1）的β亚基封闭氨基末端以及从牛脑中纯化得到的三种βγ复合物（β1γ2、β1γ3和β1γ7）可能发挥的作用。结构分析表明，每个β1亚基都有一个N-乙酰化丝氨酸，这似乎对膜结合没有贡献。由于对感光细胞膜进行蛋白酶处理或热处理均不影响Tβγ与膜的结合，所以视紫红质（或其他膜蛋白）作为接受Tβγ的锚定蛋白的可能性似乎不大。事实上，Tβγ能与磷脂大单层囊泡（LUVs）结合，其中极性头部基团对结合有强烈影响：单独的Tβγ对带负电荷的磷脂酰丝氨酸-LUVs的结合力比对中性磷脂酰胆碱（PC）-LUVs高2倍，而Tα/Tβγ复合物对磷脂酰丝氨酸-LUVs的亲和力低于对PC-LUVs的亲和力。这些结果表明：1）Tβγ与膜表面之间的离子相互作用在稳定的膜结合中起重要作用；2）三聚体状态和解离状态下，Tβγ负责结合的结构域会有所不同。我们还发现，与Tγ的COOH末端区域相对应的合成肽只有在进行异戊二烯化修饰时才会抑制Tα-Tβγ相互作用。这表明当Tβγ与Tα形成复合物时，异戊二烯基部分位于亚基之间的接触位点，而非膜结合结构域。
