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: Mine Franke, Thomas Braulke, Stephan Storch
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 288 Issue 2 Pg. 1238-49 (Jan 11 2013) ISSN: 1083-351X [Electronic] United States
• PMID: 23192343 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Protein Sorting Signals
• Arginine
• Transferases (Other Substituted Phosphate Groups)
• alpha-beta GlcNAc-1-phosphotransferase, human

Topics

• Amino Acid Sequence
• Animals
• Arginine (metabolism)
• COS Cells
• Chlorocebus aethiops
• Endoplasmic Reticulum (enzymology, metabolism)
• Golgi Apparatus (enzymology, metabolism)
• HeLa Cells
• Humans
• Microscopy, Confocal
• Microscopy, Fluorescence
• Molecular Sequence Data
• Mutation
• Protein Sorting Signals
• Protein Transport
• Sequence Homology, Amino Acid
• Transferases (Other Substituted Phosphate Groups) (chemistry, genetics, metabolism)