Role for Sit4p-dependent mitochondrial dysfunction in mediating the shortened chronological lifespan and oxidative stress sensitivity of Isc1p-deficient cells

Abstract

Saccharomyces cerevisiae cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase 2, display a shortened lifespan and an increased sensitivity to oxidative stress. A lipidomic analysis revealed specific changes in sphingolipids that accompanied the premature ageing of Isc1p-deficient cells under severe calorie restriction conditions, including a decrease of dihydrosphingosine levels and an increase of dihydro-C(26) -ceramide and phyto-C(26) -ceramide levels, the latter raising the possibility of activation of ceramide-dependent protein phosphatases. Consequently, deletion of the SIT4 gene, which encodes for the catalytic subunit of type 2A ceramide-activated protein phosphatase in yeast, abolished the premature ageing and hydrogen peroxide sensitivity of isc1Δ cells. SIT4 deletion also abolished the respiratory defects and catalase A deficiency exhibited by isc1Δ mutants. These results are consistent with catabolic derepression associated with the loss of Sit4p. The overall results show that Isc1p is an upstream regulator of Sit4p and implicate Sit4p activation in mitochondrial dysfunction leading to the shortened chronological lifespan and oxidative stress sensitivity of isc1Δ mutants.

Fig. 1

The de novo synthesis and catabolism of sphingolipids in yeast. LCFA, long chain fatty acid (C_16–18); MLCFA, medium long chain fatty acid (C_20–22); VLCFA, very long chain fatty acid (C_24–26), FFA, free fatty acid.

Fig. 2

Levels of long chain sphingoid bases during chronological aging. S. cerevisiae BY4741, isc1Δand lcb4Δ cells were grown in YPD medium to the post-diauxic phase, washed twice with H₂O and kept in H₂O at 26°C. (A) DHS; (B) PHS; (C) DHS-1-P; (D) PHS-1-P. Data are expressed as pmol of lipid/nmol lipid Pi and are means ± SD of three independent experiments.

Fig. 2

Levels of long chain sphingoid bases during chronological aging. S. cerevisiae BY4741, isc1Δand lcb4Δ cells were grown in YPD medium to the post-diauxic phase, washed twice with H₂O and kept in H₂O at 26°C. (A) DHS; (B) PHS; (C) DHS-1-P; (D) PHS-1-P. Data are expressed as pmol of lipid/nmol lipid Pi and are means ± SD of three independent experiments.

Fig. 2

Levels of long chain sphingoid bases during chronological aging. S. cerevisiae BY4741, isc1Δand lcb4Δ cells were grown in YPD medium to the post-diauxic phase, washed twice with H₂O and kept in H₂O at 26°C. (A) DHS; (B) PHS; (C) DHS-1-P; (D) PHS-1-P. Data are expressed as pmol of lipid/nmol lipid Pi and are means ± SD of three independent experiments.

Fig. 2

Levels of long chain sphingoid bases during chronological aging. S. cerevisiae BY4741, isc1Δand lcb4Δ cells were grown in YPD medium to the post-diauxic phase, washed twice with H₂O and kept in H₂O at 26°C. (A) DHS; (B) PHS; (C) DHS-1-P; (D) PHS-1-P. Data are expressed as pmol of lipid/nmol lipid Pi and are means ± SD of three independent experiments.

Fig. 3

Levels of ceramides during chronological aging. S. cerevisiae BY4741, isc1Δ and lcb4Δ cells were grown in YPD medium to the post-diauxic phase, washed twice with H₂O and kept in H₂O at 26°C. (A) total dihydroceramide (dh-Cer); (B) dihydro-C₂₆-ceramide (dh-C₂₆-Cer); (C) total phytoceramide (phyto-Cer); (D) phyto-C₂₆-ceramide (phyto-C₂₆-Cer); (E) total α–hydroxylated-phytoceramide (αHO-phyto-Cer). Data are expressed as pmol of lipid/nmol lipid Pi and are means ± SD of three independent experiments. See Tables S1–S3 for levels of individual ceramide species.

Fig. 3

Levels of ceramides during chronological aging. S. cerevisiae BY4741, isc1Δ and lcb4Δ cells were grown in YPD medium to the post-diauxic phase, washed twice with H₂O and kept in H₂O at 26°C. (A) total dihydroceramide (dh-Cer); (B) dihydro-C₂₆-ceramide (dh-C₂₆-Cer); (C) total phytoceramide (phyto-Cer); (D) phyto-C₂₆-ceramide (phyto-C₂₆-Cer); (E) total α–hydroxylated-phytoceramide (αHO-phyto-Cer). Data are expressed as pmol of lipid/nmol lipid Pi and are means ± SD of three independent experiments. See Tables S1–S3 for levels of individual ceramide species.

Fig. 3

Levels of ceramides during chronological aging. S. cerevisiae BY4741, isc1Δ and lcb4Δ cells were grown in YPD medium to the post-diauxic phase, washed twice with H₂O and kept in H₂O at 26°C. (A) total dihydroceramide (dh-Cer); (B) dihydro-C₂₆-ceramide (dh-C₂₆-Cer); (C) total phytoceramide (phyto-Cer); (D) phyto-C₂₆-ceramide (phyto-C₂₆-Cer); (E) total α–hydroxylated-phytoceramide (αHO-phyto-Cer). Data are expressed as pmol of lipid/nmol lipid Pi and are means ± SD of three independent experiments. See Tables S1–S3 for levels of individual ceramide species.

Fig. 3

Levels of ceramides during chronological aging. S. cerevisiae BY4741, isc1Δ and lcb4Δ cells were grown in YPD medium to the post-diauxic phase, washed twice with H₂O and kept in H₂O at 26°C. (A) total dihydroceramide (dh-Cer); (B) dihydro-C₂₆-ceramide (dh-C₂₆-Cer); (C) total phytoceramide (phyto-Cer); (D) phyto-C₂₆-ceramide (phyto-C₂₆-Cer); (E) total α–hydroxylated-phytoceramide (αHO-phyto-Cer). Data are expressed as pmol of lipid/nmol lipid Pi and are means ± SD of three independent experiments. See Tables S1–S3 for levels of individual ceramide species.

Fig. 3

Levels of ceramides during chronological aging. S. cerevisiae BY4741, isc1Δ and lcb4Δ cells were grown in YPD medium to the post-diauxic phase, washed twice with H₂O and kept in H₂O at 26°C. (A) total dihydroceramide (dh-Cer); (B) dihydro-C₂₆-ceramide (dh-C₂₆-Cer); (C) total phytoceramide (phyto-Cer); (D) phyto-C₂₆-ceramide (phyto-C₂₆-Cer); (E) total α–hydroxylated-phytoceramide (αHO-phyto-Cer). Data are expressed as pmol of lipid/nmol lipid Pi and are means ± SD of three independent experiments. See Tables S1–S3 for levels of individual ceramide species.

Fig. 4

SIT4 disruption suppresses the shortened chronological lifespan of isc1Δ cells. S. cerevisiae BY4741, iscΔ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to post-diauxic phase. (A) Cells were washed twice with H₂O, and kept in H₂O at 26°C. The viability was determined by standard dilution plate counts and expressed as the percentage of the colony-forming units at time 0h. (B) Protein carbonylation. Protein extracts were derivatized with DNPH and slot-blotted into a PVDF membrane. Immunodetection was performed using an anti-DNP antibody, as described in Materials and Methods. Quantitative analysis of total protein carbonyl content was performed by densitometry using data taken from the same membrane. Data are expressed as the carbonyl content ratio between cells of day 14 and cells of day 0. Values are means ± SD of three independent experiments. *p<0.05.

Fig. 4

SIT4 disruption suppresses the shortened chronological lifespan of isc1Δ cells. S. cerevisiae BY4741, iscΔ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to post-diauxic phase. (A) Cells were washed twice with H₂O, and kept in H₂O at 26°C. The viability was determined by standard dilution plate counts and expressed as the percentage of the colony-forming units at time 0h. (B) Protein carbonylation. Protein extracts were derivatized with DNPH and slot-blotted into a PVDF membrane. Immunodetection was performed using an anti-DNP antibody, as described in Materials and Methods. Quantitative analysis of total protein carbonyl content was performed by densitometry using data taken from the same membrane. Data are expressed as the carbonyl content ratio between cells of day 14 and cells of day 0. Values are means ± SD of three independent experiments. *p<0.05.

Fig. 5

SIT4 deletion suppresses hydrogen peroxide sensitivity of isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to the exponential phase (OD₆₀₀=0.6) (white bars) and exposed to 1.5 mM H₂O₂ for 30 min (black bars). (A) Cell viability was determined by standard dilution plate counts and expressed as the percentage of the colony-forming units of non-stressed cells. (B) Intracellular oxidation. Cells were labeled with the molecular probe H₂DCFDA and lysed as described in Materials and Methods. Data was normalized for protein content. (C) Protein carbonylation. Proteins were derivatized with DNPH and slot-blotted into a PVDF membrane. Immunodetection was performed using an anti-DNP antibody, as described in Materials and Methods. Quantitative analysis of total protein carbonyl content was performed by densitometry using data taken from the same membrane. Values are means ± SD of three independent experiments. *p<0.05, **p<0.01.

Fig. 5

SIT4 deletion suppresses hydrogen peroxide sensitivity of isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to the exponential phase (OD₆₀₀=0.6) (white bars) and exposed to 1.5 mM H₂O₂ for 30 min (black bars). (A) Cell viability was determined by standard dilution plate counts and expressed as the percentage of the colony-forming units of non-stressed cells. (B) Intracellular oxidation. Cells were labeled with the molecular probe H₂DCFDA and lysed as described in Materials and Methods. Data was normalized for protein content. (C) Protein carbonylation. Proteins were derivatized with DNPH and slot-blotted into a PVDF membrane. Immunodetection was performed using an anti-DNP antibody, as described in Materials and Methods. Quantitative analysis of total protein carbonyl content was performed by densitometry using data taken from the same membrane. Values are means ± SD of three independent experiments. *p<0.05, **p<0.01.

Fig. 5

SIT4 deletion suppresses hydrogen peroxide sensitivity of isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to the exponential phase (OD₆₀₀=0.6) (white bars) and exposed to 1.5 mM H₂O₂ for 30 min (black bars). (A) Cell viability was determined by standard dilution plate counts and expressed as the percentage of the colony-forming units of non-stressed cells. (B) Intracellular oxidation. Cells were labeled with the molecular probe H₂DCFDA and lysed as described in Materials and Methods. Data was normalized for protein content. (C) Protein carbonylation. Proteins were derivatized with DNPH and slot-blotted into a PVDF membrane. Immunodetection was performed using an anti-DNP antibody, as described in Materials and Methods. Quantitative analysis of total protein carbonyl content was performed by densitometry using data taken from the same membrane. Values are means ± SD of three independent experiments. *p<0.05, **p<0.01.

Fig. 6

SIT4 disruption abolishes mitochondrial dysfunctions seen in isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to exponential (log) or post-diauxic phase (PDS). (A) Oxygen consumption rates were measured as described in Materials and Methods. (B) Cytochrome C Oxidase (COX) specific activity. Cells were lysed, and enzyme activity was measured as described in Materials and Methods. (C) Cells were plated in 5-fold dilution series on glucose or glycerol as carbon source. Values are means ± SD of three independent experiments. **p<0.01.

Fig. 6

SIT4 disruption abolishes mitochondrial dysfunctions seen in isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to exponential (log) or post-diauxic phase (PDS). (A) Oxygen consumption rates were measured as described in Materials and Methods. (B) Cytochrome C Oxidase (COX) specific activity. Cells were lysed, and enzyme activity was measured as described in Materials and Methods. (C) Cells were plated in 5-fold dilution series on glucose or glycerol as carbon source. Values are means ± SD of three independent experiments. **p<0.01.

Fig. 6

SIT4 disruption abolishes mitochondrial dysfunctions seen in isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to exponential (log) or post-diauxic phase (PDS). (A) Oxygen consumption rates were measured as described in Materials and Methods. (B) Cytochrome C Oxidase (COX) specific activity. Cells were lysed, and enzyme activity was measured as described in Materials and Methods. (C) Cells were plated in 5-fold dilution series on glucose or glycerol as carbon source. Values are means ± SD of three independent experiments. **p<0.01.

Fig. 7

SIT4 disruption suppresses catalase A deficiency seen in isc1Δ cells and CTA1 overexpression partially suppresses the shortened chronological lifespan of isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to post-diauxic phase (PDS). Catalaseactivity was determined (A) spectrophotometrically or (B) detected in situ after non-denaturing polyacrylamide gel electrophoresis, using the H₂O₂/peroxidase system. (C) S. cerevisiae BY4741 and isc1Δ mutant cells transformed with pPGK-M28-I (empty vector) or pCTA1-GFP were grown in SC medium lacking uracil to post-diauxic phase. Cells were washed twice with H₂O, and kept in H₂O at 26°C. The viability was determined by standard dilution plate counts and expressed as the percentage of the colony-forming units at time 0h. (D) CTA1 overexpression increases Cta1p activity in both S. cerevisiae BY4741 and isc1Δ cells. Enzyme activity was detected as in (b). Values are means ± SD of three independent experiments. **p<0.01.

Fig. 7

SIT4 disruption suppresses catalase A deficiency seen in isc1Δ cells and CTA1 overexpression partially suppresses the shortened chronological lifespan of isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to post-diauxic phase (PDS). Catalaseactivity was determined (A) spectrophotometrically or (B) detected in situ after non-denaturing polyacrylamide gel electrophoresis, using the H₂O₂/peroxidase system. (C) S. cerevisiae BY4741 and isc1Δ mutant cells transformed with pPGK-M28-I (empty vector) or pCTA1-GFP were grown in SC medium lacking uracil to post-diauxic phase. Cells were washed twice with H₂O, and kept in H₂O at 26°C. The viability was determined by standard dilution plate counts and expressed as the percentage of the colony-forming units at time 0h. (D) CTA1 overexpression increases Cta1p activity in both S. cerevisiae BY4741 and isc1Δ cells. Enzyme activity was detected as in (b). Values are means ± SD of three independent experiments. **p<0.01.

Fig. 7

SIT4 disruption suppresses catalase A deficiency seen in isc1Δ cells and CTA1 overexpression partially suppresses the shortened chronological lifespan of isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to post-diauxic phase (PDS). Catalaseactivity was determined (A) spectrophotometrically or (B) detected in situ after non-denaturing polyacrylamide gel electrophoresis, using the H₂O₂/peroxidase system. (C) S. cerevisiae BY4741 and isc1Δ mutant cells transformed with pPGK-M28-I (empty vector) or pCTA1-GFP were grown in SC medium lacking uracil to post-diauxic phase. Cells were washed twice with H₂O, and kept in H₂O at 26°C. The viability was determined by standard dilution plate counts and expressed as the percentage of the colony-forming units at time 0h. (D) CTA1 overexpression increases Cta1p activity in both S. cerevisiae BY4741 and isc1Δ cells. Enzyme activity was detected as in (b). Values are means ± SD of three independent experiments. **p<0.01.

Fig. 7

SIT4 disruption suppresses catalase A deficiency seen in isc1Δ cells and CTA1 overexpression partially suppresses the shortened chronological lifespan of isc1Δ cells. S. cerevisiae BY4741, isc1Δ, sit4Δ and sit4Δisc1Δ mutant cells were grown in YPD medium to post-diauxic phase (PDS). Catalaseactivity was determined (A) spectrophotometrically or (B) detected in situ after non-denaturing polyacrylamide gel electrophoresis, using the H₂O₂/peroxidase system. (C) S. cerevisiae BY4741 and isc1Δ mutant cells transformed with pPGK-M28-I (empty vector) or pCTA1-GFP were grown in SC medium lacking uracil to post-diauxic phase. Cells were washed twice with H₂O, and kept in H₂O at 26°C. The viability was determined by standard dilution plate counts and expressed as the percentage of the colony-forming units at time 0h. (D) CTA1 overexpression increases Cta1p activity in both S. cerevisiae BY4741 and isc1Δ cells. Enzyme activity was detected as in (b). Values are means ± SD of three independent experiments. **p<0.01.

Fig. 8

Proposed model for Sit4p-dependent oxidative stress sensitivity and shortened chronological lifespan of isc1Δ cells. Cells lacking Isc1p display an increase in dh-C₂₆-Cer and phyto-C₂₆-Cer species, probably due to de novo biosynthesis, that activate Sit4p. Dephosphorylation of Sit4p target proteins causes mitochondrial dysfunction, leading to oxidative stress sensitivity and shortened chronological lifespan in isc1Δ cells. SIT4 deletion restores mitochondrial function of isc1Δ cells, increasing oxidative stress resistance and chronological lifespan.