Spatial Organization of Glycolytic Enzymes on Mitochondria via O-GlcNAcylation

Glucose metabolism starts with the activity of the first rate-limiting step enzyme Hexokinase (HK). In the brain, which the major energy source is glucose, HK-1 is the dominant isoform, and mostly associated with the mitochondrial outer membrane. This positioning of HK1 is critical, because it couples glucose metabolism to the energy production pathways of mitochondria. Here, we report a new molecular mechanism that regulates the mitochondrial positioning and activity of HK-1 via the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes a reversible posttranslational modification of proteins by adding a GlcNAc sugar moiety to serine and threonine residues. The catalytic activity of OGT is regulated by intracellular UDP-GlcNAc concentrations, which fluctuate proportionally in response to nutrient flux through the hexosamine pathway. In this study, We show that HK-1 is dynamically modified with O-GlcNAc at its regulatory domain. O-GlcNAcylation of HK-1 is elevated when cells were transfected with OGT or treated with TMG. We further characterized O-GlcNAc modification of HK1 recruited it on mitochondria and increases its enzymatic activity. Our findings may reveal key molecular pathways that couple neuronal metabolism to mitochondrial function via OGT, and how their dysregulation leads to neurological disorders