From bud scars to molecular insights: investigating V-ATPase function and assembly in yeast replicative aging

Abstract

Aging is a complex process that involves the progressive decline of physiological functions over time. As the global population continues to age, understanding the mechanisms underlying aging has become an important area of study. Lysosomes play a crucial role in maintaining protein quality control and degrading unneeded or damaged proteins through proteolysis. Therefore, lysosomes play a prominent role in theories of aging due to their significant role in cellular homeostasis. Interestingly, many of the hallmarks of aging are conserved between yeast and humans, highlighting the relevance of yeast as a model organism to study aging processes. One key enzyme responsible for acidifying lysosomes and lysosome-like vacuoles in yeast is the vacuolar-type H+-ATPase (V-ATPase). Despite evidence showing that lysosomes alkalinize with age, compromising their proteolytic function, little is known about the regulation of V-ATPase in aging cells. Yeast cells divide asymmetrically with each division leaving a "bud scar" that can be stained to determine replicative age. In comparing cells of distinct replicative age, we find significant decreases in V-ATPase assembly, accompanied by poor vacuolar acidification, in older cells. Remarkably, partial disassembly of the V-ATPase occurs at a relatively early age, indicating its potential as a phenotypic driver in the aging process. Reversible disassembly is controlled in part by the activity of two opposing and conserved factors, the RAVE complex and Oxr1. The RAVE complex promotes V-ATPase assembly and a rav1∆ mutant has a significantly shorter lifespan than wild-type cells; Oxr1 promotes disassembly and an oxr1∆ mutation significantly extends lifespan. These data indicate that reduced V-ATPase assembly may drive the loss of lysosome acidification with age and place the balance of V-ATPase assembly factors at the center of this process. Caloric restriction, defined as reduced calories with adequate nutrition, has been shown to extend lifespan in multiple organisms including yeast. We find that caloric restriction reverses the age-related decreases in V-ATPase assembly and vacuolar acidification in yeast as well as restoring balance of assembly factors. We investigated three conserved metabolic signaling pathways that have been linked to acidification, caloric restriction, and aging: PKA, mTORC1/S6K (TORC1/Sch9 in yeast), and AMPK (Snf1 in yeast). By utilizing non-essential nutrient mutations in these signaling pathways, we determined the impact on V-ATPase assembly during replicative aging. Mutations compromising TORC1 function were known to extend lifespan and preserved V-ATPase assembly even in older cells. In contrast, a mutation that prevents recruitment of Snf1/AMPK to vacuoles prevented V-ATPase assembly even in young cells and shortened lifespan. This study provides novel insights into the importance of V-ATPase assembly and function in the aging process and suggests novel interventions to promote health aging.