Signal transfer in the plant plasma membrane: phospholipase A(2) is regulated via an inhibitory Gα protein and a cyclophilin.
Signal transfer in the plant plasma membrane: phospholipase A(2) is regulated via an inhibitory Gα protein and a cyclophilin.
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植物质膜中的信号传递：磷脂酶 A(2) 通过抑制性 Gα 蛋白和亲环蛋白进行调节
DOI：
10.1042/bj20120793
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Conrad
中科院分区：
文献类型：
--
作者：
Heinze;Massalski;Hermann;Conrad
已知加州罂粟的质膜含有与Gα蛋白相关的磷脂酶A2，该蛋白有助于酵母糖蛋白的激活，从而诱导植物防御素的生物合成。为了了解蛋白质复合体的功能结构，我们用Gα蛋白(天然的或重组的)滴定纯化的质膜，发现临界量的这个亚基使PLA2处于低活性状态，通过激发子和GTP或通过提高Gα浓度来释放它，这可能导致Gα的寡聚，这得到了FRET(荧光共振能量转移)导向的荧光成像和半定量分裂泛素分析的支持。G-α的所有作用均被特异性抗体阻断。低G-α突变体显示PLA2活性升高，缺乏激发子对GTP的依赖刺激，但经G-α预孵育后恢复了这种能力。G-α对PLA2的抑制作用和依赖GTP的刺激作用可被肽基-脯氨酰顺式转移酶抑制剂减弱。抗G-α抗体免疫沉淀物和质膜中的亲环素被序列鉴定。我们的结论是，可溶性和靶向相关的G-α在质膜上相互作用，形成不同结构和信号放大的复合体。蛋白质折叠活性可能是传递从G-α到其目标磷脂酶A_2的构象转变所必需的。
The plasma membrane of the California poppy is known to harbour a PLA2(phospholipase A2) that is associated with the Gαprotein which facilitates its activation by a yeast glycoprotein, thereby eliciting the biosynthesis of phytoalexins. To understand the functional architecture of the protein complex, we titrated purified plasma membranes with the Gαprotein (native or recombinant) and found that critical amounts of this subunit keep PLA2in a low-activity state from which it is released either by elicitor plus GTP or by raising the Gαconcentration, which probably causes oligomerization of Gα, as supported by FRET (fluorescence resonance energy transfer)-orientated fluorescence imaging and a semiquantitative split-ubiquitin assay. All effects of Gαwere blocked by specific antibodies. A low-Gαmutant showed elevated PLA2activity and lacked the GTP-dependent stimulation by elicitor, but regained this capability after pre-incubation with Gα. The inhibition by Gαand the GTP-dependent stimulation of PLA2were diminished by inhibitors of peptidylprolylcis–transisomerases. A cyclophilin was identified by sequence in the plasma membrane and in immunoprecipitates with anti-Gαantibodies. We conclude that soluble and target-associated Gαinteract at the plasma membrane to build complexes of varying architecture and signal amplification. Protein-folding activity is probably required to convey conformational transitions from Gαto its target PLA2.