LYSET facilitates integration of both the N- and C-terminal transmembrane helices/cytoplasmic domains of GlcNAc-1-phosphotransferase.

LYSET is a recently identified Golgi transmembrane (TM) protein, and inactivating mutations in the LYSET gene phenocopy mucolipidosis II (MLII), the lysosomal storage disease caused by loss of function of GlcNAc-1-phosphotransferase αβ (GNPTαβ), which tags lysosomal hydrolases with the mannose 6-phosphate (M6P) tag for delivery to lysosomes. It is conceivable that LYSET facilitates integration of both hydrophilic TM helices (TMHs) of GNPTαβ and retain the latter in the Golgi, although this has only been directly demonstrated for the N-terminal TMH wherein a membrane-stabilized GNPTαβ variant restores lysosomal function in cells lacking LYSET. Here we show that the C-terminal TMH of GNPTαβ also contributes to LYSET-mediated Golgi retention. In addition, disease-causing patient mutations in the N-terminal TMH of GNPTαβ, which increase the hydrophilicity of the helix, are partly rescued by overexpression of LYSET. Finally, we show that a membrane-stabilized GNPTαβ variant, despite overcoming the requirement for LYSET, still requires COPI-mediated recycling via the N-terminal cytosolic domain (CD) for GNPTαβ retention and function in the Golgi.