The authors of “Glutathione depletion activates the yeast vacuolar transient receptor potential channel, Yvc1p, by reversible glutathionylation of specific cysteines” (Mol. Biol. Cell [2016] 27, 3913–3925; originally published in MBoC In Press as 10.1091/mbc.E16-05-0281) wish to make corrections to their figures. In the original version in Figure 1A, a panel of spotting (50 μM GSH, 0.5 M CaCl2) is a duplicate of the 2.5 μM GSH, 0 mM CaCl2 panel and is now replaced with the correct panel. Also, in line 5 of the legend to Figure 1, 5 and 50 μM glutathione has been corrected to 2.5 and 50 μM glutathione. In Figure 4B, the Anti GSH panel for Diamide and the Anti His Panel for H2O2 Western blots were inadvertently duplicated and are now replaced with the correct Western blot images.
These corrections do not alter the conclusion of the original figures nor of the paper. The correct versions appear below. The authors apologize for these mistakes.
The HTML and PDF versions were corrected on the Molecular Biology of the Cell website on September 18, 2019. These corrections may not appear on copies of the article that reside on other websites.
Figure 1.
Glutathione-depleted cells are more sensitive to calcium stress. (A) The S. cerevisiae WT and gsh1∆ strains were grown to exponential phase in minimal medium containing 100 μM glutathione, harvested, washed, resuspended in water, and serially diluted to give 0.1, 0.01, 0.001, and 0.0001 A600 of cells. Ten microliters of these dilutions was spotted on minimal medium containing 0.4, 0.5, and 0.6 M CaCl2 each at 2.5 and 50 μM glutathione. The photographs were taken after 2 d of incubation at 30°C. (B) gsh1∆ strain was grown in the absence of glutathione and in the presence of different concentrations of BAPTA-AM (0, 5, 10, and 25 μM). Growth curve was plotted by taking OD600nm at 4 h intervals for 16 h until the cells reached stationary phase. The bars correspond to the mean of three independent experiments ± SD. (C) Cellular DNA fragmentation assay. gsh1∆ cells grown in the absence of glutathione and in the presence of different concentrations of BAPTA-AM were harvested at 12 h, and equal OD of cells were taken and stained for DNA fragmentation with TUNEL reaction containing fluorescently tagged dUTP. Quantification of apoptotic cells; % positive cells was determined from >100 cells from different field views. Error bars correspond to the mean of three independent experiments ± SD. Statistical difference: **p < 0.01; ***p < 0.001. (D) Annexin V staining for exposition of phosphatidylserine at membrane surface. gsh1∆ cells grown in the absence of glutathione and in the presence of different concentrations of BAPTA-AM were harvested at 12 h, and equal OD of cells were taken and stained with Alexa Fluor 488–labeled Annexin V and PI. Quantitation of apoptotic cells; % of Annexin V–positive cells and dead cells; % of PI positive was done by flow-cytometric analysis. Results are represented as a histogram. The results reported are from three independent experiments. (E) Fluorescence and differential interface–contrast micrographs showing apoptotic cells as Annexin (+) PI (−) and dead cells as Annexin (+) PI (+).
Figure 4.
Yvc1p glutathionylation takes place under glutathione depletion. (A) In vitro glutathionylation analysis of Yvc1p. Purified Yvc1p was incubated with GSH (1 mM) and diamide (400 μM) in the presence and absence of cysteine modifying agents NEM and IAM and analyzed by Western blot. (B) Diamide and H2O2 increase glutathionylation of Yvc1p in vivo. Cells overexpressing Yvc1p with OD = 1.5 were treated with diamide (1 mM) and H2O2 (1.5 mM) for 20 min. After being washed, cells were lysed using glass beads, and His-tagged Yvc1p protein was purified using Ni-NTA beads and analyzed by Western blot. (C) Glutathione depletion influences the glutathionylation state of Yvc1p. WT and gsh1∆ strains overexpressing Yvc1p and transformed with ChaC1 and vector were cultured without and with 100 μM glutathione in medium. Cells were harvested at OD = 1.5. After being washed, cells were lysed using glass beads, and His-tagged Yvc1p protein was purified using Ni-NTA beads and analyzed by Western blot. Western analysis of the above experiments was carried with an equal amount of protein resolved using 10% SDS–PAGE and electroblotted on nitrocellulose membrane. The blots were probed with mouse anti-His and mouse anti-GSH primary antibodies and goat anti-mouse HRP-conjugated IgG as secondary antibody. The signal was detected using Luminata forte Western HRP substrate. The total protein expression was then quantified by densitometry analysis of protein bands. The data are expressed as the percentage protein expression compared with control (Anti HIS) expression level and are the mean ± SD of three independent experiments. Statistical difference: *, p < 0.05; **, p < 0.01; ***p < 0.001.