Supplementary File 1 Raw DIA intensity values and processed data used to identify proteins with altered stability across conditions

Hsp90 is an essential molecular chaperone that plays a key role in maintaining protein homeostasis by assisting the folding, activation, and maturation of diverse client proteins. Hsp90 functions through a dynamic conformational cycle that is coupled to ATP hydrolysis. Its ATP driven conformational cycle is finely regulated by post translational modifications and a network of co chaperones. Among these regulators, Accelerator of Hsp90 ATPase 1 (Ahsa1) is the most potent stimulator of Hsp90 ATPase activity, yet its mechanistic role in metazoans, and its potential Hsp90 independent functions, remain incompletely understood. This thesis dissects the biochemical and cellular functions of a conserved 20-residue N-terminal segment of Ahsa1, termed the intrinsic chaperone domain (ICD), and evaluates Ahsa1-dependent proteome turnover. Quantitative ATPase assays, structure guided mutagenesis, and cell based co immunoprecipitation revealed that removing the ICD accelerates Hsp90 ATPase activity in vitro and impairs stable Ahsa1–Hsp90 recruitment in cells. Surprisingly, this region has also recently been shown to contribute to an Hsp90-independent chaperone role. The work here also demonstrates that the NxNNWHW motif is also essential in the mammalian protein function, revealing key differences and similarities in yeast and metazoan protein. To probe cellular roles of Ahsa1, a novel proteome wide bioorthogonal non-canonical amino acid tagging (BONCAT) pulse chase workflow was performed to measure in cell protein degradation kinetics. BONCAT profiling showed that Ahsa1 knock down modestly destabilises a select set of proteins, whereas over expression, especially of the ICD deleted variant, reshapes the half lives of hundreds of proteins, including factors beyond the canonical Hsp90 client repertoire. These data, coupled with in vitro holdase assays, support dual Hsp90 dependent and autonomous activities for Ahsa1. The study has also uncovered previously unappreciated roles for Ahsa1 in signalling and innate immune pathways. Together, these findings expand the current understanding of co-chaperone function by revealing new structural and functional roles for Ahsa1 in proteome maintenance. This work introduces BONCAT pulse chase proteomics as a powerful tool for studying chaperone mediated proteome dynamics and advancing understanding of co chaperone biology.