On protein oxidation, lifespan and aging in Saccharomyces cerevisiae

Abstract: In this thesis, we have investigated the physiology of protein oxidation and its possible role in the aging of the yeast Saccharomyces cerevisiae. There are two ways to measure yeast aging. First, they can only divide a finite number of times even when all nutrients necessary for division are provided. This is replicative aging. Alternatively, when cells are limited with a nutrient, they exit the cell cycle and enter a G0 phase. Over time, these cells will die. This is conditional senescence or chronological aging. Thus replicative and chronological aging are two very distinct phenomena.

We demonstrated that exiting the cell cycle led to a sudden increase in protein oxidation. This increase is associated with a shift in respiratory state i.e. the amount of oxygen consumption that can be attribute to ADP phosphorylations by the ATP synthase complex is greatly reduced. Residual respiration is due to the proton conductance through the mitochondria inner membrane. In addition, we found that a mutant with a constitutive high RAS/cAMP/Protein Kinase A activity was constitutively respiring with a respiratory state close to state 4 (non-phosphorylating). This mutant clearly exerted two distinct sets of phenotypes. Some were suppressible by lowering the protein Kinase A activity as others were not. PKA independent phenotypes include a respiratory state with altered ADP phosphorylating activity. This type of respiration is often associated with high free radicals production. Indeed, this mutant also exhibited elevated level of oxygen free radicals and protein oxidation. The respiratory state deficiency was suppressed by ectopic expression of the mammalian uncoupling protein 1. This latter observation remains unexplained to date.

Finally, we developed a new technique that allowed us to visualize carbonylated proteins in situ. This technique was successfully used in yeast, bacteria and stem cells. In yeast, we showed that oxidatively damaged proteins are inherited asymmetrically at the time of mitotic cell division. This phenomenon is a hitherto unknown mechanism for defence against oxidative damage. In addition we show that the asymmetric inheritance of oxidized proteins is dependent on the presence of the Silencing Information Regulator Sir2p, a key determinant of replicative lifespan in yeast. We also found that an intact actin cytoskeleton is necessary for proper segregation of damaged proteins.