Multiple Domains of GlcNAc-1-phosphotransferase Mediate Recognition of Lysosomal Enzymes

Eline van Meel,Wang-Sik Lee,Lin Liu,Yi Qian,Balraj Doray,Stuart Kornfeld

doi:10.1074/jbc.m116.714568

Abstract

The Golgi enzyme UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous non-lysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the α subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif.

Highlights

Correct targeting of newly synthesized acid hydrolases to lysosomes is essential for this organelle to maintain its function of degrading intracellular and endocytosed material
We show that the ␥ subunit binds to the spacer region between Notch 2 and the DNA methyltransferase-associated protein (DMAP) interaction domain, and we document a variable requirement for the Man-6-P receptor homology (MRH) domain and the DMAP interaction domain of the ␥ subunit
To determine whether the residual hydrolases observed in the GNPTABϪ/Ϫ and GNPTGϪ/Ϫ cells contained any mannose 6-phosphate (Man-6-P) residues, we measured their binding to beads containing immobilized CI-Man-6-P receptors (MPRs), which exhibits high affinity binding to phosphorylated lysosomal proteins [23]

Summary

Introduction

Correct targeting of newly synthesized acid hydrolases to lysosomes is essential for this organelle to maintain its function of degrading intracellular and endocytosed material. Our findings establish that the two Notch repeats along with the DMAP interaction domain of the ␣ subunit of GlcNAc-1-phosphotransferase mediate the specific recognition of lysosomal acid hydrolases.

Results

Conclusion