Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses

To achieve system-level insights into proteome organization, regulation, and function, we developed an approach to generate complex cell pools with endogenously tagged proteins amenable to high-throughput visualization and perturbation. Pooled imaging coupled to in-situ barcode sequencing identified the subcellular localization of each HaloTag tagged protein, and subsequent ligand-induced misfolding of the library followed by single cell RNA sequencing revealed responses to spatially restricted protein misfolding. These unique datasets characterized protein quality control responses in previously uninterrogated cellular compartments, and cross-compartment analyses revealed mutually exclusive rather than collaborative responses, whereby the Heat Shock Response (HSR) is induced in some compartments and repressed in others where autophagy genes are induced. We further assign protein quality control functions of previously uncharacterized genes based on the similarity of the transcriptional responses to protein misfolding across cellular compartments. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis.