title: A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.
abstract: There are more than 600 receptor-like kinases (RLKs) inArabidopsis, 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.
authors.affiliation: From the Department of Biochemistry and.|the Biotechnology Center, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Benjamin B
authors.initials: BB
authors.lastname: Minkoff
authors.affiliation: From the Department of Biochemistry and.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Makino
authors.affiliation: From the Department of Biochemistry and.|the Biotechnology Center, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Miyoshi
authors.initials: M
authors.lastname: Haruta
authors.affiliation: From the Department of Biochemistry and.
authors.forename: Emily T
authors.initials: ET
authors.lastname: Beebe
authors.affiliation: From the Department of Biochemistry and.
authors.forename: Russell L
authors.initials: RL
authors.lastname: Wrobel
authors.affiliation: From the Department of Biochemistry and.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
authors.affiliation: From the Department of Biochemistry and msussman@wisc.edu.|the Biotechnology Center, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sussman
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008954:Models, Biological; D016296:Mutagenesis; D010770:Phosphotransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000072417:Protein Domains; D017346:Protein Serine-Threonine Kinases
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D010770:Phosphotransferases; C522131:At3g51550 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases
keywords: FERONIA; cell-free translation; enzyme mutation; lipid bilayer; membrane protein; mutagenesis in vitro; nanodiscs; plant protein kinase; receptor protein serine/threonine kinase