PROJECT SUMMARY One of the major goals of aging research is to understand how cells gradually degenerate overtime. Decades of work by molecular and cell biologicals have discovered several conserved hallmarks of cellular aging, including mitochondrial dysfunction and vacuole/lysosome defects. These aging hallmarks are usually studied one at a time and left the causes and connections between them largely unknown. What are the triggers and connections between molecular events that step-by-step culminate at the different hallmarks of aging? Without the big picture of how each protein changes in a proteome, often the best we can do is guess and try when it comes to the causes and connections between these hallmarks. To fill the gaps, we have developed and implemented a new high-throughput imaging method to systematically track the fate of each individual protein, including its expres- sion, localization, aggregation, and timing of these changes (4D fate map), during the replicative aging of budding yeast. The ongoing 4D fate mapping effort has demonstrated its value in revealing novel age-related molecular signatures and the primary causes for the well-known hallmarks of aging. A representative example from our pilot fate mapping effort is that the age-associated reduction of Tom70 is a key event of mitochondrial aging. Overexpressing Tom70 can prevent the age-associated defects in mitochondrial biogenesis and vacuole acidi- fication--two hallmarks of cellular aging. The goal of this proposal is to take a deep dive into the molecular mech- anisms of these novel Tom70 functions, which will serve as an example of using proteome fate mapping to reveal unknown causes and connections of aging hallmarks (Aim 1 and Aim 2). As our preliminary study has cleared the technical barriers, we will finish the mapping of entire yeast proteome within this grant cycle. The completion of fate mapping (Aim 3) will not only complement the other two aims by unfolding additional mechanisms that contribute to the aging of mitochondria and vacuole (Aim 1 and Aim 2) but also fill the gaps between all other aging hallmarks. This is exemplified by our preliminary results that the ongoing fate mapping provided an unex- pected mechanism of Tom70 reduction during aging. We expect to reveal additional mechanisms of Tom70 reduction and mitochondrial/vacuolar aging when fate mapping covers more proteins. Together, this project will advance both the depth and breadth of our understanding of aging and provide examples of how the fate map (Aim 3) can be used to comprehensively understand the multifactorial causes of aging hallmarks (e.g., mitochon- drial and vacuole defects in Aim 1 and Aim 2). Mining the 4D map of proteome aging by the research community will systematically unveil previous unknown mechanisms and connections between different aging hallmarks. The completion of this project will benefit researchers seeking the triggers and connections between different aging hallmarks.