The Saccharomyces cerevisiae Genes, AIM45, YGR207c/CIR1 and YOR356w/CIR2, Are Involved in Cellular Redox State Under Stress Conditions

Abstract

Mammalian electron transfer flavoproteins comprise a mitochondrial matrix heterodimer, and an electron transfer flavoprotein dehydrogenase localized in the mitochondrial inner membrane. Electrons from primary acyl-CoA dehydrogenases, of mitochondrial metabolism of fatty acids and amino acids, are transferred to the matricial heterodimer and, subsequently, to the electron transfer flavoprotein dehydrogenase, which transfers electrons to ubiquinone of the mitochondrial electron transport chain. Several evidences suggest that these proteins may convey electrons directly to molecular oxygen, yielding reactive oxygen species. In this work, we investigated phenotypes of the yeast mutants affected in the orthologous genes of the matrix heterodimer (AIM45 and YGR207c/CIR1) and of the electron transfer flavoprotein dehydrogenase (YOR356w/CIR2). The mutant strains aim45 and yor356w/cir2 displayed better growth on several non-fermentable carbon sources, which depended on the component of the electron transport chain that accepts the electrons resulting from its mitochondrial oxidation. Furthermore, upon heat shock, the mutant strains presented decreased intracellular oxidation, suggesting that these flavoproteins are a source of reactive oxygen species. Other phenotypes identified suggest that AIM45, YGR207c/CIR1 and YOR356w/CIR2 can protect cells from oxidative and heat stress, which encompass increased heat stress sensitivity, superoxide sensitivity, both only on non-fermentable carbon sources.

Keywords: Electron transfer flavoproteins (ETF); intracellular oxidation under stress conditions.; mitochondrion.

Fig. (1)

Mutants affected in genes encoding ETF and ETF-dH display higher growth rate on non-fermentable carbon sources. Cells of the parental strain (BY4741) and of mutants affected in AIM45 (aim45), YGR207c/CIR1 (cir1) and YOR356w/CIR2 (cir2) were grown overnight and diluted to OD₆₀₀=1. From this suspension, 3µL of 10^-1-10^-5 dilutions were spotted on rich media containing glucose (YPD), acetate (YPAcet), fumarate (YPFum), ethanol (YPEth), lactate (YPLact), succinate (YPSucc), pyruvate (YPPyr), malate (YPMal) or methanol (YPMeth) as sole carbon source. After 48h incubation at 30ºC, plates were photographed. Photographs represent typical results from at least three independent experiments.

Fig. (2)

Mutants affected in genes encoding ETF and ETF-dH are more sensitive to heat stress and menadione when respiring ethanol. Cells of the parental strain (BY4741) and of mutants affected in AIM45 (aim45), YGR207c/CIR1 (cir1) and YOR356w/CIR2 (cir2) were grown overnight and diluted to OD₆₀₀=1. From this suspension, 3µL of 10^-1-10^-5 dilutions were spotted on rich media containing glucose (YPD) or ethanol (YPEth) as sole carbon source andsupplemented with 3.5mM H₂O₂ or 0.025mM menadione sodium bisulfite (MBS). After 48h incubation at 30ºC or 37ºC, plates were photographed. Photographs represent typical results from at least three independent experiments.

Fig. (3)

Mutants affected in genes encoding ETF and ETF-dH display decreased intracellular oxidation upon heat shock. Cells of the parental strain (BY4741) and of mutants affected in AIM45(aim45), YGR207c/CIR1 (cir1) and YOR356w/CIR2 (cir2) were loaded with the oxidant-sensitive fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate and exposed to 50ºC for 30min. Fluorescence was recorded in each cell lysate and compared to loaded cells without exposition to 50ºC. *P < 0.05 and **P < 0.01 when compared with strain BY4741 with the same time incubation..

Fig. (4)

Altered peroxisome proliferation in cells affected in AIM45 and YGR207c/CIR1 upon heat stress and antimycin A treatment, respectively. Mutant cells affected in AIM45 (aim45), YGR207c/CIR1 (cir1) and YOR356w/CIR2 (cir2) and the correspondent parental strain (BY4741), harboring a plasmid containing the green fluorescent protein gene fused with a peroxisome-targeting signal peptide (skl), were grown overnight on YPD, ressuspended in fresh medium, and incubated at 39ºC for 6h (A) or incubated in the presence of antimycin A at 30ºC for 28h (B) and cells containing more than 2 peroxisomes were counted on a fluorescence microscope. C: bright field and GFP fluorescence representative micrographs of strains BY4741 and ygr207c/cir1 from the antimycin A incubation experiment. *P < 0.05 when compared with strain BY4741.