Post-translational Modifications of the γ-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 (␣ 2 ␤ 2 ␥ 2 ). The ␣-and ␤-subunits are catalytically active, whereas the function of the ␥-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse ␥-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed ␥-subunits were localized in the cis-Golgi apparatus. Secreted forms of ␥-subunits were detectable in media of cultured cells as well as in human serum. The ␥-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 ␥-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 ␥-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric ␥-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 ␥-subunits.

Mucolipidosis III GNPTG Missense Mutations Cause Misfolding of the γ Subunit of GlcNAc-1-Phosphotransferase

“…The WT protein showed an equal shift in migration after both endoglycosidase H f and PNGase F treatment (Fig. 1E), which is in agreement with previous observations that the γ subunit contains two high mannose-type N -linked glycans (Encarnacao, et al, 2011). Treatment of the p.G106S mutant had a similar effect, but it still exhibited a slightly slower migration.…”

“…While they do not represent residues that are directly involved in mannose binding, they are highly conserved among various species. The residue C142 was previously implicated in the formation of intramolecular disulfide bonds (Encarnacao, et al, 2011). Consistent with this report, our data show that the variant p.C142Y did not impair intermolecular disulfide linkages, as γ dimer formation was intact.…”

Intracellular lectins are involved in quality control of glycoproteins

“…65,66) GlcNAc-1-phosphotransferase preferentially transfers GlcNAc-1-phosphate to mannose on the C-arm of M8B high mannose-type glycans, and the trimming of α1,2-linked mannose from the B-arm by ER α-mannosidase I enhances the transfer of GlcNAc-1-phosphate (Fig. 3B).…”

Transport of the GlcNAc-1-phosphotransferase α/β-Subunit Precursor Protein to the Golgi Apparatus Requires a Combinatorial Sorting Motif

“…Enzymatic Deglycosylation of Proteins-For enzymatic deglycosylation of proteins total cell extracts were incubated with PNGase F or endo H for 1 h at 37°C as described previously (21).…”