Abstract

This thesis describes studies in which the yeast Saccharomyces cerevisiae was used as a model organism for unveiling the mechanisms underlying longevity regulation and
extension by genetic, dietary and pharmacological interventions. We found that a diet known as caloric restriction (CR) modulates oxidation-reduction processes and reactive oxygen species (ROS) production in yeast mitochondria, reduces the frequency of mitochondrial DNA (mtDNA) mutations, and alters the abundance and mtDNA-binding activity of mitochondrial nucleoid-associated proteins. Our findings provide evidence that these mitochondrial processes play essential roles in regulating longevity of chronologically active yeast by defining their viability following cell entry into a quiescent state. Based on these findings, we propose a hypothesis that ROS, which are mostly generated as by-products of mitochondrial respiration, play a dual role in regulating longevity of chronologically aging yeast. On the one hand, if yeast
mitochondria are unable (due to a dietary regimen) to maintain ROS concentration below a toxic threshold, ROS promote aging by oxidatively damaging certain mitochondrial
proteins and mtDNA. On the other hand, if yeast mitochondria can (due to a dietary regimen) maintain ROS concentration at a certain “optimal” level, ROS delay chronological aging. We propose that this “optimal” level of ROS is insufficient to damage cellular macromolecules but can activate certain signaling networks that extend
lifespan by increasing the abundance or activity of stress-protecting and other anti-aging proteins. In addition, studies presented in this thesis imply that mtDNA mutations do not contribute to longevity regulation in yeast grown under non-CR conditions but make important contribution to longevity regulation in yeast placed on a CR diet. The
nonreducing disaccharide trehalose has been long considered only as a reserve carbohydrate. However, recent studies in yeast suggested that this osmolyte can protect cells and cellular proteins from oxidative damage elicited by exogenously added ROS. Trehalose has been also shown to affect stability, folding and aggregation of bacterial and
firefly proteins heterologously expressed in heat-shocked yeast cells. Our investigation of how a lifespan-extending CR diet alters the metabolic history of chronologically aging yeast suggested that their longevity is programmed by the level of metabolic capacity - including trehalose biosynthesis and degradation - that yeast cells developed prior to entry into quiescence. To investigate whether trehalose homeostasis in chronologically aging yeast may play a role in longevity extension by CR, we examined how single-genedeletion mutations affecting trehalose biosynthesis and degradation impact 1) the agerelated
dynamics of changes in trehalose concentration; 2) yeast chronological lifespan under CR conditions; 3) the chronology of oxidative protein damage, intracellular ROS
level and protein aggregation; and 4) the timeline of thermal inactivation of a protein in heat-shocked yeast cells and its subsequent reactivation in yeast returned to low temperature. Our data imply that CR extends yeast chronological lifespan by altering a pattern of age-related changes in trehalose concentration.