The disease-associated hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2 β2 γ2 ) catalyzes the formation of mannose 6-phosphate residues on lysosomal enzymes required for efficient targeting to lysosomes. Using pull-down experiments and mutant subunits, we identified a potential loop-like region in the α-subunits comprising residues 535-588 and 645-698 involved in the binding to γ-subunits. The interaction is independent of the mannose 6-phosphate receptor homology domain but requires the N-terminal unstructured part of the γ-subunit consisting of residues 26-69. These studies provide new insights into structural requirements for the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.
