A Cell-free Method for Expressing and Reconstituting Membrane Proteins Enables Functional Characterization of the Plant Receptor-like Protein Kinase FERONIA [Plant Biology]

There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins means, 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 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 to other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity while the transmembrane domain is inhibitory. An ATP analog that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these—K565 and K663—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 FERONIA's protein kinase catalytic activity and show promise for future characterization of eukaryotic membrane proteins.

from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2liMWoX
via IFTTT