Post-translational Modifications of the γ-Subunit Affect Intracellular Trafficking and Complex Assembly of GlcNAc-1-phosphotransferase

Marisa Encarnação,Katrin Kollmann,Maria Trusch,Thomas Braulke,Sandra Pohl

doi:10.1074/jbc.m110.202382

Marisa Encarnação, Katrin Kollmann + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.202382

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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.

Highlights

GRK1459, FOR885, and SFB877. □S The on-line version of this article contains supplemental Figs
The data indicate the presence of high mannose-type oligosaccharides on the human ␥-subunit of GlcNAc-1-phosphotransferase
When expressed in COS-7 cells, the presence of high mannose-type oligosaccharides and the colocalization with the Golgi apparatus marker GM130 suggested that the ␥-subunits were localized in the cis-Golgi apparatus

Summary

Introduction

GRK1459, FOR885, and SFB877. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. The bovine GlcNAc-1-phosphotransferase is composed of three subunits forming a 540-kDa heterohexameric complex (␣2␤2␥2) that is encoded by two genes, GNPTAB and GNPTG (4 – 6). GNPTAB codes for the ␣/␤-subunit precursor type III membrane protein of 1265 amino acids. The highly N-glycosylated ␣/␤-subunit precursor is proteolytically cleaved into the individual subunits and contains the catalytic activity and binding sites for lysosomal enzymes (9 –11). The ␥-subunit of GlcNAc-1-phosphotransferase represents a soluble glycoprotein of 305 amino acids that forms disulfidelinked homodimers [5]. It is retained in the lumen of the cisGolgi apparatus, most likely by interactions with the integral ␣- and ␤-subunits of the GlcNAc-1-phosphotransferase complex [12, 13]. The substitution of cysteine 245 required for dimerization of ␥-subunits impaired the GlcNAc-1-phosphotransferase complex assembly

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