title: Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase.
abstract: GlcNAc-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate, a recognition marker essential for efficient transport of lysosomal hydrolases to lysosomes. The enzyme complex is composed of six subunits (α(2)β(2)γ(2)). The α- and β-subunits are catalytically active, whereas the function of the γ-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse γ-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed γ-subunits were localized in the cis-Golgi apparatus. Secreted forms of γ-subunits were detectable in media of cultured cells as well as in human serum. The γ-subunit contains two in vivo used N-glycosylation sites at positions 88 and 115, equipped with high mannose-type oligosaccharides. (35)S pulse-chase experiments and size exclusion chromatography revealed that the majority of non-glycosylated γ-subunit mutants were integrated in high molecular mass complexes, failed to exit the endoplasmic reticulum (ER), and were rapidly degraded. The substitution of cysteine 245 involved in dimerization of γ-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric γ-subunits failed, however, to associate with other GlcNAc-1-phosphotransferase subunits. The data provide evidence that assembly of the GlcNAc-1-phosphotransferase complex takes place in the ER and requires dimerization of the γ-subunits.
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.
authors.forename: Marisa
authors.initials: M
authors.lastname: Encarnação
authors.forename: Katrin
authors.initials: K
authors.lastname: Kollmann
authors.forename: Maria
authors.initials: M
authors.lastname: Trusch
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
authors.forename: Sandra
authors.initials: S
authors.lastname: Pohl
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D051379:Mice; D055503:Protein Multimerization / Q000502:physiology*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D017855:Transferases (Other Substituted Phosphate Groups); C505859:alpha-beta GlcNAc-1-phosphotransferase, human; C548056:GNPTAB protein, mouse