3 mM 4-DPS and 200 μL 1 M DTT, respectively, and was performed for each sample. The fluorescence of the cells was detected in 96-well plates (FluoroNunc, Nunc) on a fluorescence
photometer (Spectramax GeminiXS, Molecular Devices, Sunnyvale, CA), where it was possible to measure the excitation of two wavelengths, namely 395 and 465 nm, corresponding to the oxidized and the reduced form of the protein. Each sample was measured four times. The fraction of oxidized roGFP (Ox) was calculated according to the following equation: (1) It is comprised of the fluorescence of fully oxidized (Fox) or reduced (Fred) cells and the untreated sample (F), respectively. Smoothened antagonist The genes of roGFP1, roGFP1_iE and roGFP1_iL were expressed in either cytosol or ER of the P. pastoris strain X-33. The ability of the three constructs to monitor redox changes in different compartments of living yeast cells was examined through comparison of the SD of the redox ratios and the range CB-839 between the total reduction and the total oxidation of the respective roGFP (Fig. 1a–d). roGFP1 sensors are ratiometric by excitation, which means that they exhibit two excitation peaks at about 395 and 465 nm, corresponding to the neutral and the anionic chromophore forms, respectively, with a single emission peak at 510 nm (Lohman & Remington, 2008). This ratiometric behavior is
advantageous, because the redox determination is independent of the concentration of the roGFP. Fluorescence measurements were taken after the treatment of all transformants with DTT and 4-DPS as described above and compared with the fluorescence of untreated cells of the same transformants. Therefore, an external calibration was not necessary. As can be seen in Fig. 1a, the observed variability in the cytosol is low for roGFP1, but much higher for the ER-optimized constructs. In the ER, roGFP1 is fully oxidized (as observed previously by Schwarzer et al., 2007; Merksamer et al., 2008), in contrast to roGFP1_iE and roGFP_iL, which are only 45–50% oxidized (Fig. 1b). According to the wider range between
totally oxidized and totally reduced states, roGFP1_iE was chosen for the determination of the redox state in the ER (Fig. 1d). The localization of the two chosen constructs for cytosol and ER was analyzed by taking images using a confocal Clomifene laser microscope. An ER pattern was present for roGFP1_iE targeted into the ER, whereas cytosolic roGFP1 was distributed all over the cell, except for the vacuole, without showing a distinct pattern (data not shown). The reduction potential was determined using Eqn. (3) derived from the Nernst equation, and the midpoint potentials E°′roGFP for roGFP1 (−287 mV), roGFP1_iE (−236 mV) and roGFP1_iL (−229 mV), respectively (Lohman & Remington, 2008): (3) According to this calculation, the cytosol of P. pastoris X-33 has a reduction potential of −295 mV.