title: Subunit interactions of the disease-related hexameric GlcNAc-1-phosphotransferase complex.
abstract: The multimeric GlcNAc-1-phosphotransferase complex catalyzes the formation of mannose 6-phosphate recognition marker on lysosomal enzymes required for receptor-mediated targeting to lysosomes. GNPTAB and GNPTG encode the α/β-subunit precursor membrane proteins and the soluble γ-subunits, respectively. Performing extensive mutational analysis, we identified the binding regions of γ-subunits in a previously uncharacterized domain of α-subunits comprising residues 535-698, named GNPTG binding (GB) domain. Both the deletion of GB preventing γ-subunit binding and targeted deletion of GNPTG led to significant reduction in GlcNAc-1-phosphotransferase activity. We also identified cysteine 70 in α-subunits to be involved in covalent homodimerization of α-subunits which is, however, required neither for interaction with γ-subunits nor for catalytic activity of the enzyme complex. Finally, binding assays using various γ-subunit mutants revealed that residues 130-238 interact with glycosylated α-subunits suggesting a role for the mannose 6-phosphate receptor homology domain in α-subunit binding. These studies provide new insight into the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany.
authors.forename: Raffaella
authors.initials: R
authors.lastname: De Pace
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany.
authors.forename: Renata Voltolini
authors.initials: RV
authors.lastname: Velho
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany.
authors.forename: Marisa
authors.initials: M
authors.lastname: Encarnação
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Marschner
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany s.pohl@uke.de braulke@uke.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, Hamburg 20246, Germany s.pohl@uke.de braulke@uke.de.
authors.forename: Sandra
authors.initials: S
authors.lastname: Pohl
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D009154:Mutation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism*
chemical_list: D017855:Transferases (Other Substituted Phosphate Groups); C505859:alpha-beta GlcNAc-1-phosphotransferase, human; C511050:GNPTAB protein, human; C504791:GNPTG protein, human