Fig. 1.

Membrane targeting of glutathione- and H₂O₂- specific redox sensors. A, Schematic representation of palmitoylated redox sensors, Grx1-roGFP2^Palm and roGFP2-Orp1^Palm. Grx1-roGFP2 and roGFP2-Orp1 were fused with the first 29 amino acid residues of human guanine nucleotide exchange factors (GEF) which contains S-palmitoylation motif (Cys23/25/26). Palmitoylated proteins can associate with biological membranes from the cytoplasmic side. B, Grx1-roGFP2^Palm and roGFP2-Orp1^Palm were transfected into HeLa cells and protein localization was analyzed using confocal microscopy. For comparison, the data for the original cytosolic constructs are shown. Scale bars, 10 µm. C, Localizations of Grx1-roGFP2^Palm and roGFP2-Orp1^Palm were assessed by immunostaining for organelle markers; EGFR at the plasma membrane and endocytic vesicles, ABCD3 at peroxisome, phosphodiesterase (PDE) at endoplasmic reticulum, TUFM in mitochondria, LAMP1 at lysosome. Scale bars, 10 µm. Co-localizations at the plasma membrane (PM; open arrow heads) and vesicular membranes (VM; filled arrow heads) are indicated.