Cellular determinants of targeted membrane protein degradation by lysosome targeting chimeras

Targeted protein degradation is a powerful therapeutic strategy that offers benefits over canonical target inhibition. Lysosome targeting chimeras (LYTACs) harness lysosome trafficking receptors such as the cation-independent mannose-6-phosphate receptor (CI-M6PR) to direct secreted and membrane proteins to lysosomes. Their development as therapeutics would benefit from mechanistic insights into the factors that govern their activity. We conducted a genome-wide CRISPR screen to identify modulators of LYTAC-mediated membrane protein degradation. Disrupting retromer genes improved LYTAC-induced degradation by reducing the recycling of LYTAC-CI-M6PR complexes from endosomes to the plasma membrane. We identified neddylated cullin 3 as a predictive marker for LYTAC efficacy. Quantitative proteomics revealed that a significant fraction of cell-surface CI-M6PR remains occupied by endogenous M6P-modified glycoproteins. Accordingly, disruption of M6P biosynthesis enhanced the internalization of LYTAC-target complexes. Our findings inform new design strategies for LYTACs with enhanced degradation activity and elucidate the biology of CI-M6PR with implications for enzyme replacement therapies.