Starvation-induced HSC70 O-GlcNAcylation activates chaperone-mediated autophagy.

O-linked β-N-acetylglucosamine (O-GlcNAc) functions as a nutrition rheostat to mediate cellular signaling pathways. It fluctuates in response to various nutritional factors, for instance, glucose availability. Previous investigations have shown that glucose deprivation upregulates O-GlcNAcylation levels. Meanwhile, starvation also activates autophagy, in particular, chaperone-mediated autophagy (CMA). But it is unknown what signal activates CMA during starvation. In the CMA pathway, heat shock cognate 70 kDa protein (HSC70) recognizes client proteins that bear a KFERQ pentapeptide motif, and delivers them for lysosomal degradation. Herein, we show that glucose depletion increases both the affinity between HSC70 and O-GlcNAc transferase, and HSC70 O-GlcNAcylation levels. We validated that HSC70 is O-GlcNAcylated at T430 according to a previous chemoproteomic screen. We further demonstrate that O-GlcNAcylation attenuates HSC70 stability, but increases its binding with known CMA substrates, such as PKM2. We thus posit that starvation-induced HSC70 O-GlcNAcylation may activate CMA. To test this, we used label-free quantitative mass spectrometry to analyze HSC70-WT and HSC70-T430A interactome, and obtained a proteome-wide potential CMA substrate pool. By studying this dataset, we identified a new CMA substrate, Ataxin-10, a protein involved in a neurologic disorder. We then validated our model by mapping a potential KFERQ motif on Ataxin-10 and showing that HSC70-T430A decreased binding with Ataxin-10. In sum, our work suggests that CMA and O-GlcNAcylation intersect at HSC70, and starvation-induced O-GlcNAcylation of HSC70 is part of the signal that activates CMA during fasting.