Tissue-specific GlcNAcylation profiling identifies substrates in translational machinery in mushroom body contributing to olfactory learning.

GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an GlcNAc binding activity derived from OGA (OGA) with TurboID proximity labeling in , we developed an GlcNAcylation profiling tool that translates GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the GlcNAc interactome in major brain regions of and found that components of the translational machinery, particularly ribosomal subunits, were abundantly GlcNAcylated in the mushroom body of brain. Hypo-GlcNAcylation induced by ectopic expression of active OGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of GlcNAcylation in , and suggests a possibility that GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.