Transport of the GlcNAc-1-phosphotransferase ?/?-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 ?-, ?-, and ?-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 ?/?-subunit precursor protein in the Golgi apparatus. Here, we have investigated structural requirements of the PT ?/?-subunit precursor protein for its efficient export from the endoplasmic reticulum (ER). Both wild-type and a cleavage-resistant type III membrane PT ?/?-subunit precursor protein are exported whereas coexpressed separate ?- and ?-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 ?/?-subunit precursor protein in the Golgi. In the presence of a dominant negative Sar1 mutant the ER exit of the PT ?/?-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 ?/?-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.

SUBMITTER: Franke M 

PROVIDER: S-EPMC3543006 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature