Fig. 6.

Photoreversible recruitment to the plasma membrane. a AtPhyB and one of the several new AtPIF6 variants, equipped with a C-terminal CAAX prenylation motif or an N-terminal EYFP tag, respectively, were encoded on a bicistronic vector with an intervening IRES sequence and expressed in NIH-3T3 cells. b Owing to the CAAX tag, AtPhyB localizes to the plasma membrane, while the EYFP-AtPIF6 variants shuttle between cytosol and plasma membrane as a function of light. Under far-red light (750 nm), the EYFP-AtPIF6 variants exhibit cytosolic localization; under red light (650 nm), they can bind to AtPhyB and translocate to the membrane. c–e Fluorescence micrographs of NIH-3T3 cells expressing AtPhyB-CAAX and different EYFP-AtPIF6 variants under far-red light (c), after red-light exposure (d), and after additional exposure to far-red light (e). The scale bar denotes 20 µm. f The relative depletion of cytosolic EYFP fluorescence under red light compared to far-red light for the EYFP-AtPIF6 variants. Data represent mean ± SD of n ≥ 12 individual cells. g Dependence of the relative fluorescence change on the overall EYFP-AtPIF6 expression level.