Investigating the Lysosome and Plasma Membrane Systems in Protecting Cells Against Age-induced Amino Acid Toxicity

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Project Summary The incidence of age-related diseases such as Alzheimer’s disease and Parkinson’s disease continues to increase as human lifespan continues to increase. Advancements in science and healthcare have resulted in effective therapies for many diseases but treatments for these debilitating diseases remain elusive. Many studies on aging focus on the dysfunction of basic cellular processes, or the hallmarks of aging. These include mitochondrial dysfunction, abnormal nutrient signaling, telomere shortening, amongst many others. We recently found that lysosomal (vacuole in yeast) deacidification is an early event in aging and precedes mitochondrial dysfunction. Vacuole deacidification results in a loss of amino acid compartmentalization, particularly cysteine, and this results in mitochondrial dysfunction. Amino acid restriction or supplementation with iron rescued mitochondrial function. These previous findings demonstrate the importance of the vacuole in amino acid homeostasis and aging. It should be noted that vacuole deacidification is a gradual process in aging and there are likely systems that collaborate with the vacuole in maintaining cellular homeostasis. Thus, we performed a screen to determine what genes are conditionally essential upon vacuole deacidification. We uncovered genes involved in the ESCRT/MVB pathway as essential under these conditions. Furthermore, we also found several genes involved in endocytosis to be conditionally essential too. The ESCRT pathway is a cellular trafficking pathway that allows for the remodeling of the plasma membrane (PM) proteome, especially nutrient transporters. When transporters are endocytosed, the ESCRT pathway recognizes ubiquitylated transporters and directs them to the vacuole for degradation. The known function of the ESCRT pathway in conjunction with the vacuole’s role in amino acid compartmentalization raises the idea that these two systems collaborate in maintaining cellular amino acid homeostasis during the aging process. This is supported by our pilot study showing that amino acid transporters (AATs) are indeed endocytosed upon vacuole deacidification. By using the budding yeast Saccharomyces cerevisiae, we will determine how amino acid uptake and metabolite pools are affected under conditions of vacuole deacidification. Furthermore, we will elucidate the signal that originates from dysfunctional vacuoles that triggers AAT endocytosis and turnover. Finally, we will test the hypothesis that Aβ inhibits ESCRT- mediated turnover of AATs and inhibits cell growth by causing amino acid toxicity. Aging remains an active field of research but how the ESCRT pathway cooperates with the vacuole in the context of aging is understudied. Furthermore, how Aβ expression affects cellular amino acid homeostasis is understudied. The goal of this proposal is to elucidate a novel mechanism for cellular aging and increase our understanding of the pathogenesis of Alzheimer’s disease.