Systematic single-cell imaging of 5,661 proteins across yeast replicative aging reveals widespread spatial proteome reorganization, concentration changes, and interaction network remodeling during aging, with intrinsic structural features—particularly surface-exposed cysteines and lysines—predicting protein susceptibility to age-associated changes across species and providing a biophysical basis for proteome breakdown during aging.

Aging is marked by a decline in cellular functions accompanied by widespread changes in mRNA and protein abundance, yet whether aging broadly remodels subcellular protein localization and concentration—and why some proteins change while others remain stable—remains unclear. This gap matters because cellular function depends not only on expression levels but also on correct spatial organization. Using yeast replicative aging as a model, we built a robotic pipeline to enrich old cells from 5,661 strains, acquired 90 million single-cell 3D images, and applied machine learning to map proteome-wide changes in localization, concentration, and aggregation throughout aging. This age-resolved single-cell atlas uncovers widespread proteome remodeling and rewiring of protein interaction networks. Moreover, structural analysis reveals biophysical determinants of age-sensitive proteome remodeling across ages and species. Together, these results reveal a structure-encoded intrinsic principle underlying spatial proteome breakdown during aging and provide a resource to dissect mechanistic links among aging hallmarks.