S-palmitoylation stabilizes OGT and the OGT-PPP1CC complex.

O-linked β- N-acetylglucosamine ( O-GlcNAc) transferase (OGT) is the sole writer for intracellular O-GlcNAcyl7ation. It catalyzes the O-GlcNAcylation of thousands of protein substrates, but relatively little is known about the post-translational modifications that occur on OGT itself. Herein, we demonstrate that OGT is S-palmitoylated at Cys-472 and Cys-477, which is mediated by the S-acyltransferase zinc finger DHHC-type palmitoyl transferase 14 (zDHHC14) and removed by acyl protein thioesterase 2 (APT2). S-palmitoylation stabilizes OGT by shunting it away from the lysosomal chaperone-mediated autophagy (CMA) pathway, as S-palmitoylation decreases the interaction between OGT and heat shock cognate 70 kDa protein (HSC70), the CMA chaperone. Using label-free quantitative mass spectrometry, we find that S-palmitoylation increases the affinity between OGT and protein phosphatase 1 catalytic subunit gamma (PPP1CC) but not protein phosphatase 1 catalytic subunit beta (PPP1CB). We further demonstrate that S-palmitoylation of OGT augments binding to Yes-associated protein-1 (YAP), a protein that associates with PPP1CC, and subsequently enhances YAP O-GlcNAcylation. Our work uncovers S-palmitoylation of OGT and CMA-mediated degradation of lysosomal OGT, which together fine-tune the activity of key OGT complexes, such as OGT-PPP1CC, and contribute to OGT substrate selectivity.