PROJECT SUMMARY Virtually all organisms experience osmotic imbalances from their environment, which can cause rapid changes in cell volume. Hypertonic exposure causes cell shrinkage and is thought to induce protein misfolding and aggregation in vivo. Despite these proteotoxic effects, the main paradigm for osmotic stress adaptation is still focused on osmolyte production and cell volume recovery. Little is known about the mechanisms that maintain proteome integrity during osmotic stress, and no studies have investigated the identity of proteins compromised by hypertonicity. Therefore, the goal of this project is to characterize the protein quality consequences (Aim 1) and responses (Aim 2) to hypertonic exposure. The research strategy leverages targeted and systems-level technologies, including aggregation assays, genome-editing, and mass spectrometry, to examine the molecular and systems-level aspects of proteostasis during osmotic stress. In Aim 1, the applicant Kathy Le will test the hypothesis that subsets of the proteome, including nascent proteins and peroxisomal proteins, are particularly susceptible to osmotically induced aggregation. Using targeted biochemical approaches, Kathy will quantify aggregation of pulse-labeled nascent chains and the effects of translation inhibition. She will also receive individualized training in mass spectrometry techniques to profile aggregation propensity across the yeast proteome, including peroxisomal proteins. In Aim 2, Kathy will test the hypothesis that chaperone-mediated refolding and proteasomal degradation contribute to osmotic stress resistance by acting on different sets of misfolded substrates. Leveraging yeast genetics, Kathy will measure the individual stress tolerance contributions of the chaperone system and the ubiquitin-proteasome system. She will also use a combination of biochemical aggregate assays and proteomics to analyze the protein substrate pools of each system and determine the extent of their overlap. The proposed project and training plan is tailored to enable Kathy to gain new experimental skills and concepts in proteostasis, a diverse research field led by multiple experts at Stanford. Dr. Brandman (sponsor) has extensive experience studying the heat shock response and the adaptability of chaperones using cell biological tools and CRISPR libraries in yeast. Dr. Kopito (co-sponsor) is a renowned expert in studying mammalian ER-associated degradation (ERAD) and misfolded protein substrate selection, offering a complementary perspective. In summary, the strong mentoring and training opportunities at Stanford will fully prepare Kathy for an independent research career. The proposed Aims will challenge the current paradigm of osmotic stress adaptation by investigating the protein quality aspects of hypertonicity. Understanding of the mechanisms that drive protein aggregate formation, refolding, and degradation in a variety of physiological contexts is needed to elucidate general pr...