A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA
A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA
复制标题
DOI：
10.1074/jbc.m116.761981
复制
发表时间：
2017-04-07
影响因子：
4.8
通讯作者：
Sussman, Michael R.
中科院分区：
文献类型：
--
作者：
Minkoff, Benjamin B.;Makino, Shin-ichi;Sussman, Michael R.
在拟南芥中有超过600种受体样激酶（RLKs），但由于与膜蛋白表征相关的挑战，只有少数具有已知的生物学功能。植物RLK FERONIA是一种肽受体，参与植物生长调节，但对其分子作用机制知之甚少。为了研究这种酶的性质，我们使用了一种无细胞的小麦胚芽为基础的表达系统，其中编码FERONIA的mRNA与编码膜支架蛋白变体MSP 1D 1的mRNA共表达。随着脂质心磷脂的加入，这些蛋白质组装成纳米盘开始。纳米盘中的FERONIA蛋白激酶活性高于可溶性蛋白，并且与其他异源表达的蛋白激酶相当。截短实验表明，胞质跨膜结构域是必需的最大FERONIA活性，而跨膜结构域是抑制性的。与赖氨酸残基反应的ATP类似物抑制催化活性并标记了四个赖氨酸;诱变证明其中两个，Lys-565和Lys-663，在活性位点协调ATP。质谱磷酸化蛋白质组学测量进一步确定了磷酸化位点，使用磷酸化模拟诱变进行了检查。这些实验的结果是一致的模型，其中激酶介导的磷酸化的C-末端区域内是抑制性的，并调节催化活性。这些数据代表了进一步了解FERONIA的蛋白激酶催化活性的分子基础，并为真核细胞膜蛋白的未来表征显示了希望。
There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.