title: Reversible photoswitchable DRONPA-s monitors nucleocytoplasmic transport of an RNA-binding protein in transgenic plants.
abstract: Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state are novel tools for monitoring intracellular protein trafficking. A codon-optimized variant of the reversibly photoswitchable fluorescent protein DRONPA was designed for the use in transgenic Arabidopsis plants. Its codon usage is also well adapted to the mammalian codon usage. The synthetic protein, DRONPA-s, shows photochemical properties and switching behavior comparable to that of the original DRONPA from Pectiniidae both in vitro and in vivo. DRONPA-s fused to the RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein 7) under control of the endogenous AtGRP7 promoter localizes to cytoplasm, nucleoplasm and nucleolus of transgenic Arabidopsis plants. To monitor the intracellular transport dynamics of AtGRP7-DRONPA-s, we set up a single-molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that AtGRP7-DRONPA-s reaches the nucleus by carrier-mediated transport. Furthermore, photoactivation experiments showed that AtGRP7-DRONPA-s is exported from the nucleus. Thus, AtGRP7 is a nucleocytoplasmic shuttling protein. Our results show that the fluorescent marker DRONPA-s is a versatile tool to track protein transport dynamics in stably transformed plants.
authors: Department of Molecular Cell Physiology, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors: Martina
authors: M
authors: Lummer
authors: Fabian
authors: F
authors: Humpert
authors: Christian
authors: C
authors: Steuwe
authors: Katharina
authors: K
authors: Caesar
authors: Mark
authors: M
authors: Schüttpelz
authors: Markus
authors: M
authors: Sauer
authors: Dorothee
authors: D
authors: Staiger
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D017360:Arabidopsis / Q000033:anatomy & histology / Q000502:physiology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D010777:Photochemistry / Q000379:methods*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D029681:Arabidopsis Proteins; D008164:Luminescent Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C118496:GRP7 protein, Arabidopsis