title: A modified GFP facilitates counting membrane protein subunits by step-wise photobleaching in Arabidopsis.
abstract: Membrane proteins exert functions by forming oligomers or molecular complexes. Currently, step-wise photobleaching has been applied to count the fluorescently labelled subunits in plant cells, for which an accurate and reliable control is required to distinguish individual subunits and define the basal fluorescence. However, the common procedure using immobilized GFP molecules is obviously not applicable for analysis in living plant cells. Using the spatial intensity distribution analysis (SpIDA), we found that the A206K mutation reduced the dimerization of GFP molecules. Further ectopic expression of Myristoyl-GFPA206Kdriven by the endogenous AtCLC2 promoter allowed imaging of individual molecules at a low expression level. As a result, the percentage of dimers in the transgenic pCLC2::Myristoyl-mGFPA206Kline was significantly reduced in comparison to that of the pCLC2::Myristoyl-GFP line, confirming its application in defining the basal fluorescence intensity of GFP. Taken together, our results demonstrated that pCLC2::Myristoyl-mGFPA206Kcan be used as a standard control for monomer GFP, facilitating the analysis of the step-wise photobleaching of membrane proteins in Arabidopsis thaliana.
authors.affiliation: Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Song
authors.affiliation: Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Xue
authors.affiliation: Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China.
authors.forename: Yinglang
authors.initials: Y
authors.lastname: Wan
authors.affiliation: Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Deng
authors.affiliation: Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China. Electronic address: linjx@ibcas.ac.cn.
authors.forename: Jinxing
authors.initials: J
authors.lastname: Lin
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D038761:Photobleaching; D011401:Promoter Regions, Genetic / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D008565:Membrane Proteins; D049452:Green Fluorescent Proteins
keywords: Arabidopsis; Monomeric GFP; Step-wise photobleaching; Subunit counting; VA-TIRFM