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