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Michel Rigoulet

Roles of mitochondrial reactive oxygen species (ROS) in energetic homeostasis and asymmetric inheritance during cytokinesis in yeast. Michel Rigoulet. Cell Growth. Substrate. ATP ADP.Pi. Biomass. How is ATP synthesis adjusted to ATP demand to reach a compromise between growth rate

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Michel Rigoulet

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  1. Roles of mitochondrial reactive oxygen species (ROS) in energetic homeostasis and asymmetric inheritance during cytokinesis in yeast Michel Rigoulet

  2. Cell Growth Substrate ATP ADP.Pi Biomass How is ATP synthesis adjusted to ATP demand to reach a compromise between growth rate and growth yield?

  3. Stationary phase Exponential phase Transition phase Biomass (mg DW/ml) ATP demand/cell (arbitrary units) Time (hours) Enthalpic growth yield ?

  4. Catabolism Maintenance Anabolism substrate ADP + Pi biomass + O2 CO2 + H2O substrate ATP + by-products + NH4+ The growth yield depends on : 1- ATP used for cell maintenance 2- ATP synthesis yield, i.e. ATP/O ratio GROWTH YIELD DURING RESPIRATORY METABOLISM OF MICROORGANISM

  5. RELATIONSHIP BETWEEN ATP SYNTHESIS AND RESPIRATORY FLUX ON ISOLATED YEAST MITOCHONDRIA 800 ATP/O max = 1.2 600 1 ATP turnover (Jp nmol ATP/min/mg prot.) 400 0.6 200 0 0 0 300 600 State 4 State 3 Respiration (Jo nat. O/min/mg prot.) The ATP/O ratio depends on : 1- stoichiometry of H+ pump 2- H+ leak across the mitochondrial inner-membrane 3- ATP turnover

  6. MICROCALORIMETRIC ANALYSIS OF THE TRANSITION FROM GROWTH TO STATIONARY PHASE Exponential phase Transition phase Stationary phase 0.3 1 0.25 Lac X 0.2 D,L-lactate (%) Heat flux (mW/ml) 0.15 0.1 biomass (mg dw/ml) P 0.1 0.05 0 0.01 4 8 12 16 20 24 28 32 36 Time (h)

  7. The enthalpic growth yield remains constant during the transition phase RELATIONSHIP BETWEEN HEATAND BIOMASS PRODUCTION Enthalpic growth yield (YH) : heat produced by the combustion of 1g of dry weight / heat dissipated + heat produced by the combustion of 1g of dry weight 10 Transition phase YH = 0.43 8 6 Heat production (kJ / l ) 4 Exponential phase YH = 0.44 2 0 0 0.1 0.2 0.3 Biomass production (g dw / l)

  8. O2 Pyruvate, Acetate Cell Biomass (X) Lactate CO2, H2O NH3 Heat (Q) E.R.= 0.98 E.R.= 1.02 X Ac 70 - X Pyr Lac - 60 Pyr Ac Lac - 50 Q Q - 40 Energy content (kJ /g dw) - 30 20 - - 10 0 Early exponential growth phase Transition growth phase ENTHALPY BALANCE DURING GROWTH

  9. OXIDATIVE PHOSPHORYLATION REGIME OF YEAST CELLS DURING LACTATE-LIMITED GROWTH G r o w t h p h ase Ea r ly s t at i ona r y Transition e xpon e n t ia l Jo 318 ± 22 223 ± 39 63 ± 14 b a sal ( n a t . O/ m i n/ mg dw ) Jo 135 ± 16 99 ± 7 35 ± 3 T E T ( n a t . O/ m i n/ mg dw ) Jo 443 ± 18 345 ± 27 123 ± 12 C l CCP ( n a t . O /m in /mg d w ) Jo /Jo 3.3 ± 0.2 3.5 ± 0.2 3.5 ± 0.2 C l CCP T E T ( Jo – Jo ) / ( Jo - Jo ) 59 ± 4 % 50 ± 6 % 32 ± 3 % b a sal T E T C l CCP T E T x100

  10. MITOCHONDRIAL ACTIVITIES IN YEAST CELLS THROUGHOUT THE CULTURE PERIOD

  11. RELATIONSHIP BETWEEN RESPIRATORY CAPACITY , CYTOCHROME CONTENT AND CITRATE SYNTHASE OF BATCH GROWN CELLS A B Stationary Transition Early 80 700 c + c1 60 500 Cytochrome content (pmol / mg dw) JOmax (nat O / mg dw) 40 b 300 20 a + a3 100 0 0 0 50 100 150 200 250 0 100 200 300 400 Citrate synthase activity (mU) Citrate synthase activity (mU)

  12. sampling On-line display (heat measurements) • Biomass • Substrate • By-products dQ/dt (µW) time Peristaltic pump Heat sink (28°C) Water bath (28°C) Water saturated air (28°C) Chamber Enthalpic balance and enthalpic growth yield measurements

  13. Alternative methods : 1/ Amount of substrate oxidized per g dry weight during a generation time Disadvantages :dependent on : - the substrate (lactate, glycerol, ethanol) - the degree of its combustion 2/ Amount of oxygen consumed per g dry weight during a generation time : QO2/µ

  14. Relationship between QO2/µ and the enthalpic growth yield

  15. Growth yield homeostasis under respiratory conditions

  16. Tpk 1,2,3 Bcy1 Cap Cyr1 Adenylate cyclase cAMP cAMP Bcy1 cAMP The Ras/cAMP pathway GDP GTP cdc25 Ras1,2 Ras1,2 Ira1,2 Pde1;2 ATP AMP Tpk 1,2,3

  17. Activation of the Ras/cAMP pathway GDP GTP cdc25 Ras1,2 Ras1,2 (Ras2Val19) Ira1,2 Tpk 1,2,3 Bcy1 Cap Cyr1 Adénylate cyclase Pde1;2 cAMP ATP AMP cAMP Bcy1 Tpk 1,2,3 cAMP Inhibition of the Ras/cAMP pathway Ras2∆

  18. Cytochrome content and respiratory capacity of yeast mutants affected in the ras/PKA pathway 200 200 c+c1 b 150 150 a+a3 % variation of respiration compared to wild-type %variation of cytochromes compared to wild-type 100 100 50 50 0 0 -50 -50 ras2val19 ras2 ira1ira2 bcy1 ira1ira2 bcy1 ras2 ras2val19

  19. GDP GTP cdc25 Ras1,2 Ras1,2 Ira1,2 Tpk 1,2,3 Bcy1 Cap Cyr1 Adénylate cyclase cAMP cAMP Bcy1 cAMP ? Tpk 1,2,3 ? Pde1;2 ATP AMP ?

  20. Respiratory growth is altered in the absence of the PKA tpk3p WT ∆Tpk3 Growth of wild type () and ∆tpk3 () cells. Cells were grown aerobically in minimal medium containing 0.2% (w/v) D, L-lactate. Growth was measured at 600nm.

  21. This is due to a decrease in mitochondrial content within the cells

  22. aa3 b cc1 Respiratory growth is altered in the absence of the PKA tpk3p and there is an important decrease in cytochrome c amount

  23. NADH, H+ NAD+ H+ c e- Q III IV II Succinate O2 NADH, H+ NAD+ H+ Fumarate H+ H2O ADP+Pi ATP+H2O The quinones are much more reduced on mitochondria isolated from the PKA(tpk3)-deficient cells

  24. Yeast mitochondrial respiratory chain O2.- NADH, H+ NAD+ H+ c e- Q III IV II Succinate O2 NADH, H+ NAD+ H+ Fumarate H+ H2O ADP+Pi O2.- ATP+H2O

  25. ROS production is drastically increased in mitochondria isolated from ∆Tpk3 mutant ROS : deleterious molecules AND true intracellular second messengers

  26. Energetic parameters are restored in the PKA deficient cells in the presence of an antioxydant wild type () and ∆tpk3 () cells in the presence of an anti-oxidant.

  27. Yeast growth on 0.6% glucose Substrate concentration time

  28. ROS level regulates the HAP2,3,4,5 nuclear transcription factor activity HAP LacZ pCYC1 2µ

  29. catalase SOD1 H2O O2.- H2O2 NADH, H+ NAD+ H+ c e- Q III IV II Succinate O2 NADH, H+ NAD+ H+ Fumarate H+ H2O O2.- ADP+Pi ATP+H2O SOD2 H2O2 H2O catalase Sites of ROS production

  30. Intermembranal space ROS are involved in the mitochondria to nucleus signaling through HAP2,3,4,5. SOD1 SOD2 Respiratory rates as a function of growth in wild type, ∆tpk3 and ∆tpk3 overexpressing SOD cells. Wild type (), ∆tpk3 (), ∆tpk3-SOD1 () and ∆tpk3-SOD2 ()

  31. HAP4 SH SH HAP4cys A B SH H + NAC + NAC Dhap4 Dhap4 Dtpk3 Dhap4 Dhap4 Dtpk3 Dhap4 Dhap4 Dtpk3 Dhap4 Dhap4 Dtpk3 + pHAP4cys + pHAP4

  32. Conclusions QO2/µ µ JO2 Growth yield homeostasis

  33. ? Upstream signal ? Mitochondrial biogenesis + HAP Tpk P Respiratory chain O2.- SOD2 H2O2 H2O catalase

  34. ? Upstream signal ? Mitochondrial biogenesis + HAP Tpk Cytosol/nucleus? P Respiratory chain O2.- O2.- O2.- O2.- O2.- SOD2 H2O2 H2O2 H2O catalase H2O2 H2O H2O2 H2O2

  35. Level of damaged proteins as a function of replicative age 8 Average number of bud scars 6 4 2 Protein carbonylation level Fraction Calcofluor (chitine) Age

  36. Asymmetric distribution of damaged proteins during cytokinesis +Paraquat Oxidation signal (pixel intensity) 2 1

  37. Distribution of mitochondria, ROS and damaged proteins during yeast cytokinesis DHE Bright Field DiOC6 Merge Bright Field Carbonyls Por1p

  38. Laboratory of Cellular Bioenergetics IBGC-CNRS University of Bordeaux 2 out Louis Casteilla Bertrand Daignan-Fornier Anne Galinier In Nicole Avéret Odile Bunoust Cyrille Chevtzoff Anne Devin Arnaud Mourier

  39. Loss of homeostasis uncoupling Overactivation of Ras/cAMPcascade

  40. HAP4 overexpression abrogates ROS induced decrease • in mitochondrial biogenesis.

  41. ROS regulate the activity of the transcription factors HAP2,3,4,5 • Activity of the transcription factors HAP2,3,4,5. The activity of the transcription factors • HAP2,3,4,5 was assessed with a widely used reporter gene, pCYC1-lacZ

  42. -100 Lac 0.2% Lac 2% -100 -80 - 80 biomass -60 - 60 Energy content (kJ / g dw) Energy content(kJ / g dw) Pyr Lac Biomass -40 - 40 Biomass Pyr biomass Lac Ac Pyr Lac Lac -20 Pyr - 20 Ac Q Q Q Q 0 0 Input Input Output Input Output Input Output Output - cAMP + 3mM cAMP - cAMP + 3mM cAMP EFFECT OF cAMP AND LACTATE CONCENTRATION ON THE ENTHALPY BALANCE OF BATCH GROWN OL556 Loss of homeostasis when the Ras/cAMP pathway is overactivated

  43. FUNCTIONAL PROPERTIES OF MITOCHONDRIA FROM OL556 CELLS GROWN IN THE PRESENCE OR ABSENCE OF cAMP Identical mitochondria (energetic properties) Increase in the amount of mitochondria per cell

  44. Replication potential Medium life span Maximal life span

  45. CONCLUSION This work suggests that ROS act as a sensor of the mitochondrial functional state and that over a threshold, they signal to the nucleus through regulation of the activity of transcription factor(s). In addition, it is clear that the site of ROS production (compartmentalization) and the concentration of ROS generation are important factors in determining the physiological actions and effects of ROS in the regulation of mitochondrial biogenesis. Such a down-regulation of mitochondrial biogenesis when mitochondrial alterations lead to increased ROS production could be seen as a mitochondria quality-control process. Further studies will be necessary to unveil the mechanisms by which ROS are able to decrease the activity of the HAP2,3,4,5 complex.

  46. P P Ways to adjust ATP synthesis : Kinetic : cAMP dependent phosphorylation of complexes I and IV Changes in the amount of mitochondria per cell cAMP

  47. 3/ Homeostasis LOSS QO2/µ µ JO2

  48. This adjustment goes through a modulation of mitochondrial content during growth

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