title: Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein.
abstract: Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, United States.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: Transmembrane Protein Center, University of Wisconsin-Madison, United States.
authors.forename: Shin-Ichi
authors.initials: SI
authors.lastname: Makino
authors.affiliation: Transmembrane Protein Center, University of Wisconsin-Madison, United States.
authors.forename: Emily T
authors.initials: ET
authors.lastname: Beebe
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, United States.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Urano
authors.affiliation: Transmembrane Protein Center, University of Wisconsin-Madison, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Aceti
authors.affiliation: Transmembrane Protein Center, University of Wisconsin-Madison, United States.
authors.forename: Tina M
authors.initials: TM
authors.lastname: Misenheimer
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, United States.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Peters
authors.affiliation: Transmembrane Protein Center, University of Wisconsin-Madison, United States.
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, United States; Department of Pharmacology, University of North Carolina at Chapel Hill, United States. Electronic address: alan_jones@unc.edu.
authors.forename: Alan M
authors.initials: AM
authors.lastname: Jones
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D020710:RGS Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D029681:Arabidopsis Proteins; D020710:RGS Proteins; C477755:RGS1 protein, Arabidopsis
keywords: 7-Transmembrane protein; Arabidopsis regulator of G signaling protein 1 (AtRGS1); In vitro translation; Membrane scaffold protein 1D1; Nanodiscs