title: Transport of the GlcNAc-1-phosphotransferase α/β-subunit precursor protein to the Golgi apparatus requires a combinatorial sorting motif.
abstract: The Golgi-resident N-acetylglucosamine-1-phosphotransferase (PT) complex is composed of two α-, β-, and γ-subunits and represents the key enzyme for the biosynthesis of mannose 6-phosphate recognition marker on soluble lysosomal proteins. Mutations in the PT complex cause the lysosomal storage diseases mucolipidosis II and III. A prerequisite for the enzymatic activity is the site-1 protease-mediated cleavage of the PT α/β-subunit precursor protein in the Golgi apparatus. Here, we have investigated structural requirements of the PT α/β-subunit precursor protein for its efficient export from the endoplasmic reticulum (ER). Both wild-type and a cleavage-resistant type III membrane PT α/β-subunit precursor protein are exported whereas coexpressed separate α- and β-subunits failed to reach the cis-Golgi compartment. Mutational analyses revealed combinatorial, non-exchangeable dileucine and dibasic motifs located in a defined sequence context in the cytosolic N- and C-terminal domains that are required for efficient ER exit and subsequent proteolytic activation of the α/β-subunit precursor protein in the Golgi. In the presence of a dominant negative Sar1 mutant the ER exit of the PT α/β-subunit precursor protein is inhibited indicating its transport in coat protein complex II-coated vesicles. Expression studies of missense mutations identified in mucolipidosis III patients that alter amino acids in the N- and C-terminal domains demonstrated that the substitution of a lysine residue in close proximity to the dileucine sorting motif impaired ER-Golgi transport and subsequent activation of the PT α/β-subunit precursor protein. The data suggest that the oligomeric type III membrane protein PT complex requires a combinatorial sorting motif that forms a tertiary epitope to be recognized by distinct sites within the coat protein complex II machinery.
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: Mine
authors.initials: M
authors.lastname: Franke
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
authors.forename: Stephan
authors.initials: S
authors.lastname: Storch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D021382:Protein Sorting Signals; D001120:Arginine; D017855:Transferases (Other Substituted Phosphate Groups); C505859:alpha-beta GlcNAc-1-phosphotransferase, human