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Reversible Photoswitchable DRONPA‐s Monitors Nucleocytoplasmic Transport of an RNA‐Binding Protein in Transgenic Plants
Author(s) -
Lummer Martina,
Humpert Fabian,
Steuwe Christian,
Caesar Katharina,
Schüttpelz Mark,
Sauer Markus,
Staiger Dorothee
Publication year - 2011
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/j.1600-0854.2011.01180.x
Subject(s) - green fluorescent protein , arabidopsis thaliana , biology , arabidopsis , protein subcellular localization prediction , microbiology and biotechnology , bimolecular fluorescence complementation , nucleoplasm , biophysics , biochemistry , cytoplasm , nucleolus , gene , mutant
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 At GRP7 ( 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 At GRP7‐DRONPA‐s, we set up a single‐molecule sensitive confocal fluorescence microscope. Fluorescence recovery after selective photoswitching experiments revealed that At GRP7‐DRONPA‐s reaches the nucleus by carrier‐mediated transport. Furthermore, photoactivation experiments showed that At GRP7‐DRONPA‐s is exported from the nucleus. Thus, At GRP7 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.