The glycosyltransferases of the mammalian Golgi complex must recycle between the stacked cisternae of that organelle to maintain their proper steady-state localization. This trafficking is mediated by COPI-coated vesicles, but how the glycosyltransferases are incorporated into these transport vesicles is poorly understood. Here we show that the N-terminal cytoplasmic tails (N-tails) of a number of Golgi glycosyltransferases which share a ϕ-(K/R)-X-L-X-(K/R) sequence bind directly to the δ- and ζ-subunits of COPI. Mutations of this N-tail motif impair binding to the COPI subunits, leading to mislocalization of the transferases to lysosomes. The physiological importance of these interactions is illustrated by mucolipidosis III patients with missense mutations in the N-tail of GlcNAc-1-phosphotransferase that cause the transferase to be rapidly degraded in lysosomes. These studies establish that direct binding of the N-tails of mammalian Golgi glycosyltransferases with COPI subunits is essential for recycling within the Golgi.

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...

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

The Golgi-localized, gamma-ear containing, ADP-ribosylation factorbinding proteins (GGAs 1, 2, and 3) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) at the Golgi and play a role, along with adaptor protein complex 1 (AP-1), in the sorting of newly synthesized lysosomal hydrolases to the endolysosomal system. However, the relative importance of the two types of coat proteins in this process is still unclear. Here, we report that inactivation of all three GGA genes in HeLa cells decreased the sorting efficiency of cathepsin D from 97% to 73% relative to wild-type, with marked redistribution of the cation-independent MPR from peripheral punctae to the trans-Golgi network. In comparison, GNPTAB À/À HeLa cells with complete inactivation of the mannose 6-phosphate pathway sorted only 20% of the cathepsin D. We conclude that the residual sorting of cathepsin D in the GGA triple-knockout cells is mediated by AP-1. A critical step in the trafficking of newly synthesized acid hydrolases to lysosomes occurs at the trans-Golgi network (TGN) where mannose 6-phosphate receptors (MPRs) bind the acid hydrolases via their mannose 6phosphate (M-6-P) tags. The receptor-ligand complexes are then incorporated into clathrin-coated vesicles (CCVs), followed by transport to the endosome/ lysosome compartment [1]. Key to the assembly of the CCVs are two coat proteins, the Golgi-localized, gamma-ear containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein complex 1 (AP-1), both of which bind clathrin and the MPRs [2,3]. A number of studies in mammalian cells have used RNA-interference (RNA-i) to knock down the various GGAs to determine the role of each GGA in acid hydrolase trafficking to lysosomes. These studies have indicated that all three GGAs play a role in this process [4-6]. In addition, triple-knockdown of the GGAs reduced the sorting efficiency of the lysosomal enzyme, cathepsin D, from 80% in control cells compared to 68% in the cells treated with siRNA targeting the three GGAs [6]. Presumably, the residual sorting is mediated by AP-1. A limitation of the RNA-i experiments is that the

UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-PT) is an α2β2γ2 hexamer that mediates the initial step in the formation of the mannose 6-phosphate targeting signal on newly synthesized lysosomal acid hydrolases. The GNPTAB gene encodes the 1256 amino acid long α/β precursor which is normally cleaved at K928 in the early Golgi by Site-1 protease (S1P). Here, we show that removal of the so-called `spacer-1´ domain (residues 86–322) results in cleavage almost exclusively at a second S1P consensus sequence located upstream of K928. In addition, GlcNAc-1-PT lacking spacer-1 exhibits enhanced phosphorylation of several non-lysosomal glycoproteins, while the phosphorylation of lysosomal acid hydrolases is not altered. In view of these effects on the maturation and function of GlcNAc-1-PT, we suggest renaming `spacer-1´ the `regulatory-1´ domain.