These two sequences were flanked by SbfI and SfiI restriction sites, and separated in between by two nonidentical FauI restriction

sites. The three roGFPs were amplified by PCR, adding the respective FauI sites. These constructs were then ligated between the KAR2 leader and the HDEL sequences, and introduced into the same pPuzzle vector as that used for the cytosolic expression. The integration locus for the ER constructs was the 5′ region of the P. pastoris enolase gene. The plasmid containing the gene PDI1 (encoding protein disulfide isomerase; Inan et al., 2006) was generated by PCR using P. pastoris genomic DNA as a template and SbfI and SfiI as restriction sites. The gene was cloned into a pPuzzle vector containing the Zeocin resistance marker, and was expressed under the control of the GAP1 promoter. The vector was integrated into the native PDI1 gene locus AZD6244 of the P. pastoris genome after linearization in the respective sequence. Electrocompetent P. pastoris host strains were transformed using a BioRad Minipulser. Conditions for the pulsing included a cuvette with a 2-mm gap, a charging voltage of 2000 V and a pulse length of 4 ms. After 2-h regeneration on YPD (per liter: 20 g yeast extract, 10 g soy peptone, 20 g glucose), cells were cultivated for 48 h and at 30 °C on YPD-agar this website plates (per liter: 20 g yeast extract, 10 g soy peptone, 20 g glucose, 20 g agar-agar) containing 25 μg mL−1

Zeocin or 100 μg mL−1 Hygromycin (both Invivogen), respectively. Shake-flask experiments were carried out in 100-mL shake flasks incubating at 28 °C at 170 r.p.m. For each strain, 12–15 individual clones were used to inoculate 10 mL of freshly prepared minimal medium. The medium used in these experiments was M2 minimal medium containing per liter: 20 g of glucose, 20 g of citric acid, 3.15 g of (NH4)2HPO4, 0.03 g of CaCl2·2H2O, 0.8 g of KCl, 0.5 g of MgSO4·7H2O,

2 mL of biotin (0.2 g L−1) and 1.5 mL of trace salts stock solution. The pH was set to 5.0 with 5 M KOH solution. Trace salts stock solution contained per liter: 6.0 g of CuSO4·5H2O, Erastin research buy 0.08 g of NaI, 3.0 g of MnSO4·H2O, 0.2 g of Na2MoO4·2H2O, 0.02 g of H3BO3, 0.5 g of CoCl2, 20.0 g of ZnCl2, 5.0 g of FeSO4·7H2O and 5.0 mL of H2SO4 (95–98% w/w). A protocol for the determination of the redox state using rxYFP in S. cerevisiae (Ostergaard et al., 2004) served as a template for the establishment of a redox-measuring procedure in living P. pastoris cells. The culture (840 μL) with an OD of approximately 30 was used for determination of the redox ratio. Redox measurements in the cytosol were performed with and without addition of the cell-solubilizing agent digitonin. Comparison of both experiments yielded the same results; therefore, further experiments were performed without digitonin. For the ER, digitonin was not added to the cells, because it would lead to a whole-cell lysis, which was not desirable in this case.