AbstractThe pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.