N-acetylglucosamine transferase is an integral component of a kinesin-directed mitochondrial trafficking complex.

Trafficking kinesin proteins (TRAKs) 1 and 2 are kinesin-associated proteins proposed to function in excitable tissues as adaptors in anterograde trafficking of cargoes including mitochondria. They are known to associate with N-acetylglucosamine transferase and the mitochondrial rho GTPase, Miro. We used confocal imaging, Förster resonance energy transfer and immunoprecipitations to investigate association between TRAKs1/2, N-acetylglucosamine transferase, the prototypic kinesin-1, KIF5C, and Miro. We demonstrate that in COS-7 cells, N-acetylglucosamine transferase, KIF5C and TRAKs1/2 co-distribute. Förster resonance energy transfer was observed between N-acetylglucosamine transferase and TRAKs1/2. Despite co-distributing with KIF5C and immunoprecipitations demonstrating a TRAK1/2, N-acetylglucosamine transferase and KIF5C ternary complex, no Förster resonance energy transfer was detected between N-acetylglucosamine transferase and KIF5C. KIF5C, N-acetylglucosamine transferase, TRAKs1/2 and Miro formed a quaternary complex. The presence of N-acteylglucosamine transferase partially prevented redistribution of mitochondria induced by trafficking proteins 1/2 and KIF5C. TRAK2 was a substrate for N-acetylglucosamine transferase with TRAK2 (S562) identified as a site of O-N-acetylglucosamine modification. These findings substantiate trafficking kinesin proteins as scaffolds for the formation of a multi-component complex involved in anterograde trafficking of mitochondria. They further suggest that O-glycosylation may regulate complex formation.