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 (?(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.

SUBMITTER: Encarnacao M 

PROVIDER: S-EPMC3037643 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature