Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase.

TY - JOUR

T1 - Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase.

AU - Encarnação, Marisa

AU - Kollmann, Katrin

AU - Trusch, Maria

AU - Braulke, Thomas

AU - Pohl, Sandra

PY - 2011

Y1 - 2011

N2 - 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.

AB - 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.

M3 - SCORING: Zeitschriftenaufsatz

VL - 286

SP - 5311

EP - 5318

JO - J BIOL CHEM

JF - J BIOL CHEM

SN - 0021-9258

IS - 7

M1 - 7

ER -