Mechanosensor-mediated Hsp70 phosphorylation orchestrates the landscape of the heat shock response

Heat shock protein 70 is an evolutionary conserved molecular chaperone responsible for the protein quality control functions. It is involved in many critical cellular processes, including folding protein ‘clients’, modulation of protein-protein interactions, and transport of proteins across membranes. Hsp70s are critical for maintaining cell viability in response to a large variety of cellular stresses. Perturbation of the proteostasis network is implicated in many diseases ranging from cancer and neurodegeneration to genetic disorders. Hsp70s are highly modified at the post-translational level. All these modifications together are referred to as the “chaperone code. These modifications fine-tune chaperone function, altering chaperone activity, localization, and selectivity. Understanding the regulation of these modifications will provide new insights into the protein folding process and characterize the direct interplay between chaperones and major signal transduction pathways. This thesis investigates a critical post-translational modification (PTM) site on yeast Heat Shock Protein 70 (Hsp70) that undergoes phosphorylation during heat shock response. Here, we focus on threonine 492 (T492), a highly conserved residue on Hsp70, which is conserved across all domains of life. Elucidating its upstream regulation and downstream effects. Yeast cells respond rapidly to heat stress by activating multiple protective mechanisms to maintain proteostasis. These include Hsf1 and Msn2/4-mediated transcriptional activation, cell integrity signaling, stress-induced bimolecular condensate formation and resolution, and protein translation inhibition. However, these pathways' rapid activation and coordination have remained poorly understood. Our findings reveal that heat-induced membrane stretch is detected by the Mechanosensor Mid2, triggering rapid phosphorylation of the cytosolic Yeast Hsp70 at T492. This phosphorylation event has several crucial downstream effects, which include altered interactome, altered dynamics of P-body resolution, maintenance of translational fidelity, amplification of the cell-wall integrity pathway, proper activation of heat-shock response, and regulation of clients Bck1 and Edc3. These results provide a comprehensive, unified theory of the global yeast shock response mediated by the Hsp70 chaperone code.