The DMAP interaction domain of UDP-GlcNAc:lysosomal enzyme
            <i>N</i>
            -acetylglucosamine-1-phosphotransferase is a substrate recognition module

Qian, Yi; Flanagan-Steet, Heather; Meel, Eline van; Steet, Richard; Kornfeld, Stuart

doi:10.1073/pnas.1308453110

Cited by 32 publications

(38 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…With regard to the role of the ␣DMAP interaction domain, it should be noted that we previously reported that the K732N and L785W missense mutations in this domain were defective in phosphorylating purified porcine CathD in vitro, and the K732N mutant was impaired in correcting the phenotypic abnormalities observed in a zebrafish model of ML II (13). Therefore, it was surprising that the ⌬DMAP mutant phosphorylated CathD quite well in the current transfection studies while exhibiting defective phosphorylation of several other lysosomal hydrolases.…”

Section: Discussionmentioning

confidence: 73%

“…␣-GalA was a generous gift from Amicus Therapeutics (Cranbury, NJ), whereas ␣-iduronidase was kindly provided by William Canfield (Genzyme, Boston, MA). GST and GST fusions were expressed in and purified from Escherichia coli BL21 (RIL) cells (Agilent Technologies), and pulldown assays were performed with the purified lysosomal enzymes exactly as described (13).…”

Section: Methodsmentioning

confidence: 99%

“…Support for this hypothesis has come from studies of the consequences of missense mutations in Notch 1 and the DMAP interaction domain found in patients with the autosomal recessive lysosomal storage disorders mucolipidosis II and III (10,13). These mutations were shown to impair lysosomal enzyme phosphorylation without altering the catalytic activity of the transferase toward the simple sugar ␣-methyl D-mannoside (␣MM).…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Meel¹,

Lee²,

Liu³

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The Golgi enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an ␣ 2 ␤ 2 ␥ 2 hexamer, 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 nonlysosomal 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 ␣ 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 ␥-binding site on the ␣ subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the ␥ 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.Correct targeting of newly synthesized acid hydrolases to lysosomes is essential for this organelle to maintain its function of degrading intracellular and endocytosed material. In higher eukaryotes, this process is mediated by the mannose 6-phosphate (Man-6-P) 5 recognition system whereby the newly synthesized acid hydrolases acquire Man-6-P residues in the Golgi that serve as high affinity ligands for binding to Man-6-P receptors (MPRs) in the trans-Golgi network and subsequent transport to the endo-lysosomal system (1). The initial and most critical step in the generation of the Man-6-P tag is mediated by the Golgi enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase). This enzyme binds selectively to conformation-dependent protein determinants in the 60 or so lysosomal acid hydrolases and transfers GlcNAc-1-P from UDP-GlcNAc to mannose residues on high mannose-type N-linked glycans of the hydrolases (2). The GlcNAc is subsequently excised by a second Golgi enzyme ("uncovering enzyme") to generate the high affinity Man-6-P ligand (3).GlcNAc-1-phosphotransferase is an ␣ 2 ␤ 2 ␥ 2 hexamer that is encoded by two genes (4 -7). The GNPTAB gene encodes the ␣ and ␤ subunits, whereas the GNPTG gene encodes the ␥ subunit. Enzyme kinetic studies have indicated that the ␣ and ␤ subunits specifically bind lysosomal acid hydrolases and mediate the catalytic function of the enzyme (8, 9). The ␥ subunit enhances the rate of GlcNAc-P transfer to a subset of the acid hydrolases without substantially altering the binding to these acceptors. Consistent with this, analysis of the level of mannose phosphorylation of the acid hydrolases in the brain of mice lacking the ␥ subunit, as estimated by the extent of binding to a cation-independent (CI)-MPR affinity resin, indicated that about one-third of the acid hydrol...

show abstract

Section: Discussionmentioning

confidence: 73%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Meel¹,

Lee²,

Liu³

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…GNPTAB-V5/His in pcDNA6 (kind gift from W. Canfield, Genzyme, Cambridge, MA; see ref. 24) was modified by quick-change sitedirected mutagenesis (Stratagene) to generate K4Q, S15Y, and S399F phosphotransferase αβ subunits. See SI Materials and Methods for the primer sequences.…”

Section: Methodsmentioning

confidence: 99%

“…Confocal immunofluorescence microscopy was performed as described in ref. 24. See the SI Materials and Methods for more details.…”

Section: Methodsmentioning

confidence: 99%

Mislocalization of phosphotransferase as a cause of mucolipidosis III αβ

Meel

Qian

Kornfeld

2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The lysosomal storage disorder mucolipidosis III αβ is caused by mutations in the αβ subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase). This Golgi-localized enzyme mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases, and loss of function results in impaired lysosomal targeting of these acid hydrolases and decreased lysosomal degradation. Here we show that two patient missense mutations, Lys4Gln and Ser15Tyr, in the N-terminal cytoplasmic tail of the α subunit of phosphotransferase impair retention of the catalytically active enzyme in the Golgi complex. This results in mistargeting of the mutant phosphotransferases to lysosomes, where they are degraded, or to the cell surface and release into the medium. The finding that mislocalization of active phosphotransferase is the basis for mucolipidosis III αβ in a subset of patients shows the importance of single residues in the cytoplasmic tail of a Golgi-resident protein for localization to this compartment.

show abstract

Identification of the interaction domains between α‐ and γ‐subunits of GlcNAc‐1‐phosphotransferase

et al. 2016

View full text Add to dashboard Cite

The disease-associated hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α β γ ) catalyzes the formation of mannose 6-phosphate residues on lysosomal enzymes required for efficient targeting to lysosomes. Using pull-down experiments and mutant subunits, we identified a potential loop-like region in the α-subunits comprising residues 535-588 and 645-698 involved in the binding to γ-subunits. The interaction is independent of the mannose 6-phosphate receptor homology domain but requires the N-terminal unstructured part of the γ-subunit consisting of residues 26-69. These studies provide new insights into structural requirements for the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.

show abstract

The DMAP interaction domain of UDP-GlcNAc:lysosomal enzyme N -acetylglucosamine-1-phosphotransferase is a substrate recognition module

Cited by 32 publications

References 25 publications

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

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

Mislocalization of phosphotransferase as a cause of mucolipidosis III αβ

Identification of the interaction domains between α‐ and γ‐subunits of GlcNAc‐1‐phosphotransferase

Contact Info

Product

Resources

About