Lack of a functional Mpc pyr Lack of a functional Mpc pyr acetate metabolism Ivan Orlandi 1,2 *, Damiano Pe 1 SYSBIO Centre for Systems Biology Milan SYSBIO Centre for Systems Biology Milan 2 Dipartimento di Biotecnologie e Bioscienze, Università degli Stu In the field of aging-related research, the yeast chronological lifespan model offers the opportunity to study th maximum survival period of a population of non-dividing cells in postmitotic stationary phase. Viability over time is intimately coupled to metabolic processes, in particular to energy metabolism and nutrient availability . Nutrien Depending on nutrient availability cells receive signals which induce them to proliferate or to enter in a quiescen regulation for the cellular survivability. In this context, mitochondria play an active and pivotal role. These org aging, including the coupling of electron transport to ATP synthesis, modulation of mitochondrial membrane p aging, including the coupling of electron transport to ATP synthesis, modulation of mitochondrial membrane p mitochondrial metabolic pathways depends on a fine tuning of the metabolite fluxes between cytosol and mit essential process which provides the organelle with one of the main substrates of both catabolic and anabolic pa Mpc complex, which is essential for the pyruvate uptake in the mitochondrial matrix (2, 3). MPC is a heterocom mitochondrial membrane. Yeast mutants lacking Mpc1 showed severe defects in mitochondrial pyruvate uptake. MPC1 inactivation shortens CLS and affects extracellular ethanol and a 100 MPC1 inactivation shortens CLS and affects extracellular ethanol and a B A C 120 16 18 60 80 al (%) 80 100 (mM) wt mpc1 10 12 14 M) BY4741 (wt) mpc1 ∆ ∆ 20 40 Surviva 40 60 Ethanol 4 6 8 Acetate (mM 0 20 0 3 6 9 12 15 18 21 Days 0 20 Days 0 1 2 3 4 0 2 4 0 Days Days Lack of Mpc1 leads to an increase of intra- and extracellular pyruvate 50 A B C 10 Lack of Mpc1 leads to an increase of intra- and extracellular pyruvate dehydrogenase activity 30 40 50 g prot) wt mpc1 6 8 10 uvate (mM) wt mpc1 3 4 vate (mM) wt mpc1 ∆ ∆ ∆ 10 20 30 Adh (U/mg 2 4 6 Extracellular Pyru 1 2 ntracellular Pyruv 0 Day Exp 0 1 0 E Days 0 In Days Exp 0 1 2 3. Exp 0 1 2 3. (A) Intra- and (B) extracellular pyruvate concentrations were measured for both wt and mpc1 ∆ cultures during exp harvested and Adh (C), Ald4 (D) and Ald6 (E) enzymatic activities were measured. Error bars are the standard deviatio mpc1∆ ∆ ∆ ∆ cells show an enhanced glyoxylate-requiring gluconeogenesis mpc1∆ ∆ ∆ ∆ cells show an enhanced glyoxylate-requiring gluconeogenesis A B 120 140 wt 80 100 120 U/mg prot) wt mpc1 ∆ 20 40 60 Icl1 (mU Ald4,6 0 Days Exp 0 1 2 3. Ald4,6 (A). Scheme of metabolic pathways allo (A). Scheme of metabolic pathways allo Ald4/6: acetaldehyde dehydrogenase 4/ activities were measured for both cultur exponential phase, day 0, diauxic shift. In three replicates. In conclusion... Summing up our results, it can be proposed tha pyruvate uptake in the mitochondrial matrix an pathways to provide acetyl-CoA. During the p pathways to provide acetyl-CoA. During the p gluconeogenic metabolism while the respiration extracellular environment. References: 1. Longo VD, Shadel GS, Kaeberlein M, Kennedy B. (2012), Replicative and chronological aging in Saccharomyces cerevisiae. Cell 2. Bricker DK, Taylor EB, Schell JC, Orsak T, Boutron A, Chen YC, Cox JE, Cardon CM, Van Vranken JG, Dephoure N, Redin C, Bo yeast, Drosophila, and humans. Science 337:96-100. 3. Herzig S, Raemy E, Montessuit S, Veuthey JL, Zamboni N, Westermann B, Kunji ER, Martinou JC., Identification and functional e ruvate carrier influences cytosolic ruvate carrier influences cytosolic m and chronological aging ellegrino Coppola 2 and Marina Vai 1,2 no, Università di Milano-Bicocca, 20126 Milano, Italy no, Università di Milano-Bicocca, 20126 Milano, Italy udi di Milano-Bicocca, 20126 Milano, Italy; *E-mail: ivan.orlandi@unimib.it. he aging process of postmitotic quiescent mammalian cells. Chronological lifespan (CLS) is the mean and is defined as the ability to resume mitotic growth upon return to rich fresh medium (1). The process of aging nts provide the energy and molecular components for cellular homeostasis and influence cellular longevity . nt state. This later condition characterizes the chronological aging in yeast and it requires a proper metabolic ganelles compartmentalize various redox processes which are essential for establishing the rate of cellular potential and maintenance of cellular homeostasis of reactive oxygen species (ROS). The efficiency of the potential and maintenance of cellular homeostasis of reactive oxygen species (ROS). The efficiency of the tochondria. Among them, the transport of pyruvate, the end product of glycolysis, into mitochondria is an athways. In Saccharomyces. cerevisiae it has been identified the mitochondrial pyruvate carrier, namely the mplex formed by two proteins, Mpc1 and Mpc2, associated to form an ~150-kilodalton complex in the inner .and impaired pyruvate metabolism (3). acetic acid levels acetic acid levels BY4741 (wt) and mpc1∆ mutant cells were grown in minimal medium (with a fourfold excess of supplements) containing 2% glucose and followed up to stationary phase. (A) CLS of wt and mpc1∆ mutant cells. At every time-point, viability was determined by counting CFUs on YEPD wt mpc1 ∆ At every time-point, viability was determined by counting CFUs on YEPD plates. 72 h after the diauxic shift (Day 3), was considered the first age- point. Error bars are the standard deviation of three replicates. Bar charts of ethanol (B) and acetic acid (C) concentrations measured in the of ethanol (B) and acetic acid (C) concentrations measured in the medium of both cultures at the indicated time-points. Day 0, diauxic shift. Days 0 1 2 3 4 Days e and an enhancement of cytosolic acetaldehyde 250 140 D E e and an enhancement of cytosolic acetaldehyde 150 200 250 mg prot) wt mpc1 80 100 120 140 U/mg prot) wt mpc1 ∆ ∆ 50 100 150 Ald6 (mU/m 40 60 80 Ald4 (mU s 0 Days 0 20 Days Exp 0 1 2 3. Exp 0 1 2 3. 2 3. ponential growth (Exp.) and at the indicated time-points. Day 0, diauxic shift. In parallel, cell samples were on of three replicates. s and a reduction of mitochondrial respiration s and a reduction of mitochondrial respiration C D 140 160 wt 12 wt 80 100 120 140 U/mg prot) wt mpc1 8 nsumption wt mpc1 10 6 cells/sec) ∆ ∆ 20 40 60 80 Pck1 (mU 4 Oxygen con (picomoli/1 0 20 Days Exp 0 1 2 3. 0 Days Exp 0 1 2 3. owing ethanol and acetate utilization. Only relevant reactions are shown. Adh2: alcohol dehydrogenase 2, owing ethanol and acetate utilization. Only relevant reactions are shown. Adh2: alcohol dehydrogenase 2, /6, Icl1: isocitrate lyase 1, Pck1: phosphoenolpyruvate carboxykinase 1. Icl1 (B) and Pck1 (C) enzymatic res of wt and mpc1∆ chronological aging cells grown on 2% glucose at the indicated time-points. Exp., n parallel, cellular respiration of the same cells was monitored (D). Error bars are the standard deviation of at mpc1∆ cells rewires the acetate metabolism to overcome the impairment of nd this reassessment seems to be based on an enhancement of cytosolic post diauxic phase acetate only in part is channeled towards the cytosolic post diauxic phase acetate only in part is channeled towards the cytosolic n is reduced and a considerable amounts of acetic acid accumulates in the l Metabolism 16:18-31. oudina S, Gygi SP, Brivet M, Thummel CS, Rutter J. (2012), A mitochondrial pyruvate carrier required for pyruvate uptake in expression of the mitochondrial pyruvate carrier. Science 337:93-96 (2012).