Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport

GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels similar to 60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are alpha 2 beta 2 gamma 2 hexamers in which the core catalytic alpha- and beta-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-l-phosphotransferase function and related diseases.

Automated summary

In this study, the structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB, was determined and the catalytic domain and putative donor substrate-binding site were identified. The functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Furthermore, we found that the homodimer formed by DmGNPTAB is evolutionarily conserved and disrupting the dimer interface affects the maturation and activity of human GNPTAB.