Cloning and direct G-protein regulation of phospholipase D from tobacco
Cloning and direct G-protein regulation of phospholipase D from tobacco
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DOI：
10.1016/s1388-1981(00)00182-7
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发表时间：
2001-02-26
影响因子：
4.8
通讯作者：
Saalbach, G
中科院分区：
文献类型：
--
作者：
Lein, W;Saalbach, G
磷脂酶D（PLD）和异源三聚体G蛋白参与植物细胞质膜上的信号转导途径。有证据表明PLD作用于CT蛋白的下游，但尚未显示特定成员的直接相互作用。本研究从烟草中克隆了PLD cDNA，并在细菌中以GST融合蛋白的形式表达，重组蛋白经谷胱甘肽亲和层析纯化。它的酶特性鉴定它是一种α型PLD。以类似的方式分离来自烟草的G蛋白的α亚基。这两种蛋白质在生物化学测定中是功能性的。当G蛋白被包括在PLD测定中时，观察到PLD活性的强烈剂量依赖性抑制。不同的对照蛋白没有表现出这种抑制作用。当GST-NtGP α 1通过与GTP γ S孵育而被激活时，抑制活性大大降低。这些结果提供了植物中异源三聚体G蛋白α亚基直接调节PLD α的第一个指示。(C)2001 Elsevier Science B. V.保留所有权利。
Phospholipase D (PLD) and heterotrimeric G-proteins are involved in plant signal transduction pathways at the plasma membrane. There is evidence suggesting that PLD acts downstream from CT-proteins, but a direct interaction of specific members has not been shown. In the present paper, a PLD cDNA clone was isolated from tobacco, expressed as a GST fusion in bacteria, and the recombinant protein was purified by glutathione affinity. Its enzymatic properties identified it as an alpha -type PLD. The alpha -subunit of a G-protein from tobacco was isolated in a similar way. Both proteins were functional in biochemical assays. When the G-protein was included in the PLD assay, a strong dosage-dependent inhibition of the PLD activity was observed. Different control proteins did not exhibit this inhibitory effect. When GST-NtGP alpha1 was activated by incubation with GTP gammaS the inhibitory activity was greatly reduced. These results provide a first indication for a direct regulation of PLD alpha by a heterotrimeric G-protein alpha -subunit in plants. (C) 2001 Elsevier Science B.V. All rights reserved.