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Decarbonylated cyclophilin A Cpr1 protein protects Saccharomyces cerevisiae KNU5377Y when exposed to stress induced by menadione

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Cell Stress and Chaperones Aims and scope

Abstract

Cyclophilins are conserved cis–trans peptidyl-prolyl isomerase that are implicated in protein folding and function as molecular chaperones. The accumulation of Cpr1 protein to menadione in Saccharomyces cerevisiae KNU5377Y suggests a possibility that this protein may participate in the mechanism of stress tolerance. Stress response of S. cerevisiae KNU5377Y cpr1Δ mutant strain was investigated in the presence of menadione (MD). The growth ability of the strain was confirmed in an oxidant-supplemented medium, and a relationship was established between diminishing levels of cell rescue enzymes and MD sensitivity. The results demonstrate the significant effect of CPR1 disruption in the cellular growth rate, cell viability and morphology, and redox state in the presence of MD and suggest the possible role of Cpr1p in acquiring sensitivity to MD and its physiological role in cellular stress tolerance. The in vivo importance of Cpr1p for antioxidant-mediated reactive oxygen species (ROS) neutralization and chaperone-mediated protein folding was confirmed by analyzing the expression changes of a variety of cell rescue proteins in a CPR1-disrupted strain. The cpr1Δ to the exogenous MD showed reduced expression level of antioxidant enzymes, molecular chaperones, and metabolic enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH)- or adenosine triphosphate (ATP)-generating systems. More importantly, it was shown that cpr1Δ mutant caused imbalance in the cellular redox homeostasis and increased ROS levels in the cytosol as well as mitochondria and elevated iron concentrations. As a result of excess ROS production, the cpr1Δ mutant provoked an increase in oxidative damage and a reduction in antioxidant activity and free radical scavenger ability. However, there was no difference in the stress responses between the wild-type and the cpr1Δ mutant strains derived from S. cerevisiae BY4741 as a control strain under the same stress. Unlike BY4741, KNU5377Y Cpr1 protein was decarbonylated during MD stress. Decarbonylation of Cpr1 protein in KNU5377Y strain seems to be caused by a rapid and efficient gene expression program via stress response factors Hsf1, Yap1, and Msn2. Hence, the decarbonylated Cpr1 protein may be critical in cellular redox homeostasis and may be a potential chaperone to menadione.

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Acknowledgments

This research was supported by a grant (#2009-0072715) from the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education, Science and Technology and by a grant (20070301-034-021) from BioGreen 21 Program, Rural Development Administration.

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  1. Department of Microbiology, Kyungpook National University, Daegu, 702-701, Republic of Korea

    Il-Sup Kim & Ingnyol Jin

  2. Department of Biology, Kyungpook National University, Daegu, 702-701, Republic of Korea

    Il-Sup Kim & Ho-Sung Yoon

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Correspondence to Il-Sup Kim.

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Fig. S1

Antioxidant enzyme activity. Relative enzyme activity of glutathione reductase (GR) (a), glutathione peroxide (GPX) (b), glucose-6-phosphate dehydrogenase (G6PDH) (c), and isocitrate dehydrogenase (IDH) (d) was calculated (JPEG 187 kb)

Fig. S2

Cpr1 expression, stress sensitivity, and redox state analysis in hsp104 mutant from KNU5377Y. a Cpr1 expression was analyzed via immunoblotting analysis with anti-Cpr1 antibody. Tubulin (Tub) was used a standard control. N, untreated cells; S, 0.4 mM MD-treated cells. b Mid-log yeast cells were streaked onto YPD agar plate supplemented with 0.05 mM MD. WT, wild-type cells; hsp104Δ, hsp104 mutant cells. c Measurement of in vivo molecular oxidation. DCF (cytosol) fluorescences were measured in yeast cells, exposed to 0.4 mM MD for 1 h. Fluorescence images were obtained under microscopy; With, MD-treated cells; Without, MD-untreated cells (JPEG 115 kb)

Fig. S3

Transcriptional level, cell viability, and redox state of HSF1 under MD stress. a Transcriptional level of HSF1 was carried out with semi-RT-PCR from total RNA in wild-type (HSF1+/+) and hsf1 mutant (HSF1+/−) from diploid KNU5377Y and BY4743 (EUROSCARF). mRNA transcript of PDA1 was used as a housekeeping control. N, MD (0.4 mM)-untreated cells; S, MD-treated cells. b Mid-log phase cells were exposed to a variety of MD concentration (0.0–0.8 mM), serially diluted to 10−5, and then dispensed onto YPD agar plate. Cell viability was calculated as counting colony. Wild-type cells (HSF+/+) (filled square) and hsf1 mutant (HSF1+/−) cells (filled circle) from KNU5377Y3; wild-type cells (HSF+/+) (filled triangle) and hsf1 mutant (HSF1+/−) cells (filled inverted triangle) from BY4743. c Mid-log phase yeast cells were exposed to 0.4 mM MD for 1 h at 30°C with shaking (160 rpm). Cellular ROS levels were analyzed using light microscope with oxidant-sensitive probe DCFHDA as described in “Materials and methods”. (JPEG 77 kb)

Fig. S4

Growth rate under normal condition. Growth kinetics was measured at 600 nm using spectrophotometer. Initial optical density is adjusted to 0.1. kwt (filled square), kc1 (filled circle), bwt (filled triangle), and bc1 (filled inverted triangle) (JPEG 77 kb)

Fig. S5

Cell viability of S. cerevisiae BY4741 in the presence of MD. Yeast cells grown at 30°C for 18 h were inoculated in fresh YPD medium (initial optical density A600 = 0.15), cultured for about 5 h at 30°C until the absorbance reached a value of 1.2–1.5 and then exposed to MD for 1 h with shaking (160 rpm). Yeast cells with and without MD were serially diluted to 10–5 and 100 μl of the 10−5-diluted solutions was dispensed onto YPD agar plate. After 2 days of incubation, CFU was counted (a) and the plate was photographed (b). For spotting assay, 5 μl of diluted solutions was spotted onto YPD agar plate (c) (JPEG 151 kb)

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Kim, IS., Jin, I. & Yoon, HS. Decarbonylated cyclophilin A Cpr1 protein protects Saccharomyces cerevisiae KNU5377Y when exposed to stress induced by menadione. Cell Stress and Chaperones 16, 1–14 (2011). https://doi.org/10.1007/s12192-010-0215-9

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