Abstract

Mechanisms Underlying Regulation of Yeast Longevity by Genetic and Pharmacological Interventions that Alter Mitochondrial Membrane Lipidome and Remodel Mitochondrial Respiratory Supercomplexes

Olivia Roseline Koupaki, M.Sc.

Recent studies in our laboratory demonstrated that in chronologically aging yeast grown under CR conditions LCA, a natural anti-aging compound, alters the age-related dynamics of changes in mitochondrial abundance and morphology, respiration, membrane potential, and ROS production.

Cardiolipin (CL), a dimeric glycerophospholipid that is synthesized and almost exclusively localized in the inner mitochondrial membrane, has been shown to modulate mitochondria-governed processes whose dysfunction underlies aging and age-related pathologies. Phosphatidylethanolamine (PE) is another glycerophospholipid that is almost exclusively synthesized in the inner mitochondrial membrane, from which it is then distributed to various other cellular membranes. Hence, it is likely that the synthesis and stability of CL and, perhaps, PE in the inner mitochondrial membrane are important targets of longevity-extending and health-improving interventions.

Because of the plausible importance of mitochondrially synthesized CL and PE in the longevity-extending effect of LCA, other graduate students in our laboratory elucidated how mutations eliminating nucleus-encoded mitochondrial proteins involved in the synthesis of CL and PE within the inner mitochondrial membrane influence the lifespan-extending efficacy of LCA in chronologically aging yeast grown under CR conditions.

The results of this genetic analysis suggested that the synthesis of both these membrane lipids in mitochondria plays an essential role in the ability of LCA to extend longevity of yeast placed on a CR diet. All these findings prompted us to elucidate how LCA influences the composition of mitochondrial membrane lipids in chronologically aging yeast grown under CR conditions.

To attain this objective, in experiments described in my thesis we used mass spectrometry (MS)-based lipidomics to elucidate the effect of LCA on the repertoire and quantities of membrane lipids in mitochondria that were purified from wild-type (WT) strain and from various long- and short-lived mutant strains impaired in different aspects of CL and PE metabolism.

By correlating the effects of LCA on the age-related dynamics of changes in the composition and quantities of membrane lipids in mitochondria of these strains to the effects of this anti-aging compound on their chronological lifespan, we concluded that under CR conditions LCA extends yeast longevity by remodeling the composition of mitochondrial membrane lipids and thereby modulating longevity-defining processes confined to and governed by mitochondria.

Specifically, findings described in my thesis strongly suggest that LCA extends longevity of WT yeast by (1) elevating the level of phosphatidylserine (PS; a precursor for the synthesis of PE in mitochondria) in the mitochondrial membrane, thereby enhancing its positive effect on longevity-defining processes in this membrane; (2) reducing the level of PE in the mitochondrial membrane, thereby weakening its negative effect on longevity-defining processes in this membrane; and (3) proportionally decreasing the levels of PE and CL in the mitochondrial membrane, thereby increasing PS/CL and PS/PE ratios but maintaining PE/CL ratio of mitochondrial membrane lipids and causing some longevity-extending changes in this membrane.

It is important to emphasize that these LCA-induced alterations in mitochondrial membrane lipids can satisfactorily explain the observed implications of LCA treatment on mitochondrial structure and function, including (1) the ability of LCA to cause dramatic changes in the length and curvature of the inner mitochondrial membrane; and (2) the ability of LCA to activate protein machines involved in mitochondrial respiration, the maintenance of mitochondrial membrane potential, ROS production in mitochondria and mitochondrial fusion.

Based on our recent findings and data of other researchers working in the field of mitochondrial biology, we hypothesized that, by altering the level of CL and other glycerophospholipids within the mitochondrial membrane, LCA could modulate the stoichiometry, composition and/or functional state of respiratory supercomplexes (respirasomes) in the inner mitochondrial membrane. To test the validity of this hypothesis, in studies described in my thesis we used a multistep method for recovery of intact respiratory complexes and supercomplexes from purified yeast mitochondria, their subsequent first-dimension electrophoretic separation using so-called blue-native gel electrophoresis (BN-PAGE), their resolution into individual protein components with the help of denaturing Tricine-SDS-PAGE, and the mass spectrometry (MS)-based identification of each of these individual protein components.

Findings described in my thesis validate our hypothesis. Specifically, these findings revealed several ways of rearranging respiratory supercomplexes in the inner mitochondrial membrane of cells exhibiting altered mitochondrial membrane lipidome in response to LCA treatment or genetic manipulations impairing the synthesis of CL and other glycerophospholipids within the inner membrane of mitochondria. First, by altering the level of CL and other glycerophospholipids synthesized and residing in the inner mitochondrial membrane, LCA modulates the abundance of several major respiratory supercomplexes (respirasomes) in this membrane. Second, LCA- and genetic manipulations-driven changes in the inner mitochondrial membrane lipidome cause a recruitment of a number of new mitochondrial protein components, not previously known for being permanently associated with the ETC, into the remodeled respirasomes. Importantly, many of the proteins newly recruited into the remodeled respirasomes are known for their essential roles in mitochondria-confined processes that define longevity.