SUMOylation targets O-GlcNAcase to chaperone-mediated autophagy.

O-GlcNAcase (OGA) is the sole eraser for the intracellular O-GlcNAc. OGA has many roles in distinct biological processes, such as cancer and embryonic stem cells, but its precise regulatory mechanism is far from being understood. Herein, we studied the small ubiquitin-like modifier (SUMO) modification of OGA and found that OGA is SUMOylated at K358. SUMOylation targets OGA to the chaperone-mediated autophagy (CMA) pathway, which shunts client proteins to the lysosome for degradation. We demonstrate that SUMOylation increases the association between OGA and the heat shock cognate protein 70 (HSC70), the CMA chaperone, and facilitates OGA further degradation. We further mapped a SUMO-interacting motif (SIM) (VLIFD, aa. 195-199) on HSC70. Notably, HSC70-SIM is essential for affinity with other CMA client proteins, such as pyruvate kinase M2. We thus posit that the SIM of HSC70 binds SUMOylated client proteins in a lock-and-key manner to confer substrate selectivity during CMA. To further test our hypothesis, we used label-free quantitative mass spectrometry to study the HSC70-SIM mutant interactome and generated a proteome-wide SUMO-mediated CMA client pool. We then validated this model by studying YEATS domain-containing two from the protein pool and demonstrated that YEATS domain-containing two is SUMOylated at K592, targeting it to CMA. Our work uncovers the SUMO-SIM interaction as a fundamental mechanism governing CMA substrate selectivity and identifies a potential CMA client proteome to deepen our understanding of its pathophysiological relevance.