A FRET method for investigating dimer/monomer status and conformation of the UVR8 photoreceptor

Liao, X., Zhang, B. , Blatt, M. R. and Jenkins, G. I. (2018) A FRET method for investigating dimer/monomer status and conformation of the UVR8 photoreceptor. Photochemical and Photobiological Sciences, (doi:10.1039/C8PP00489G) (Early Online Publication)

[img]
Preview
Text
174622.pdf - Published Version
Available under License Creative Commons Attribution.

3MB

Abstract

The photoreceptor UVR8 has a pivotal role in mediating plant responses to UV-B wavelengths. Dimeric UVR8 dissociates into monomers following UV-B photoreception, and there is evidence that this process is accompanied by conformational changes that may facilitate interaction of UVR8 with other proteins to initiate signaling. Hence monitoring UVR8 dimer/monomer status and conformation is key to understanding UVR8 action. Here we have used Fluorescence Resonance Energy Transfer (FRET) to study these processes in both wild-type and mutant UVR8 proteins in vivo. UVR8 was fused to GFP and mCherry at the C- and N-termini, respectively and both the FRET efficiency and loss of GFP fluorescence after photobleaching were measured. In addition, measurements were made for UVR8 fused to either GFP or mCherry to eliminate intra-molecular FRET signals. The results indicate that dissociation of UVR8 dimer to monomer principally accounts for the loss of FRET signal for wild-type UVR8 and there is little evidence of a contribution from conformational change in vivo. Examination of plants expressing UVR8W285F and UVR8D96N,D107N are consistent with these mutant proteins being constitutively dimeric and monomeric, respectively. The methods employed here will be valuable for monitoring UVR8 dimer/monomer status in vivo in relation to signaling, and will facilitate characterization of dimer/monomer status and conformation of further UVR8 mutants.

Item Type:Articles
Status:Early Online Publication
Refereed:Yes
Glasgow Author(s) Enlighten ID:Jenkins, Professor Gareth and Blatt, Professor Michael and Zhang, Dr Ben and Liao, Xinyang
Authors: Liao, X., Zhang, B., Blatt, M. R., and Jenkins, G. I.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Photochemical and Photobiological Sciences
Publisher:Royal Society of Chemistry
ISSN:1474-905X
ISSN (Online):1474-9092
Published Online:04 December 2018
Copyright Holders:Copyright © 2018 The Royal Society of Chemistry and Owner Societies
First Published:First published in Photochemical and Photobiological Sciences 2018
Publisher Policy:Reproduced under a Creative Commons License

University Staff: Request a correction | Enlighten Editors: Update this record

Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
721491SAURs in stomatal regulation New Title: 15 NSFBIO SAUR regulation of stomatal apertureMichael BlattBiotechnology and Biological Sciences Research Council (BBSRC)BB/P011586/1RI MOLECULAR CELL & SYSTEMS BIOLOGY
719021Synthesis of Microcompartments in Plants for Enhanced Carbon FixationMichael BlattBiotechnology and Biological Sciences Research Council (BBSRC)BB/N01832X/1RI MOLECULAR CELL & SYSTEMS BIOLOGY