Possible pathways for lysosomal enzyme delivery.

Abstract

Immunogold double-labeling and ultrathin cryosections were used to compare the subcellular distribution of albumin, mannose 6-phosphate receptor (MPR), galactosyltransferase, and the lysosomal enzymes cathepsin D, beta-hexosaminidase, and alpha-glucosidase in Hep Gz cells. MPR and lysosomal enzymes were found throughout the stack of Golgi cisternae and in a trans-Golgi reticulum (TGR) of smooth-surfaced tubules with coated buds and vesicles. The trans-Golgi orientation of TGR was ascertained by the co-localization with galactosyltransferase. MPR was particularly abundant in TGR and CURL, the compartment of uncoupling receptors and ligands. Both TGR and CURL also contained lysosomal enzymes, but endogenous albumin was detected in TGR only. The coated buds on TGR tubules contained MPR, lysosomal enzymes, as well as albumin.MPR and lysosomal enzymes were also found in coated pits of the plasma membrane. CURL tubules seemed to give rise to smooth vesicles, often of the multivesicular body type. In CURL, the enzymes were found in the lumina of the smooth vesicles while MPR prevailed in the tubules. These observations suggest a role of CURL in transport of lysosomal enzymes to lysosomes.When the cells were treated with the lysosomotropic amine primaquine, binding of anti-MPR to the cells in culture was reduced by half. Immunocytochemistry showed that MPR accumulated in TGR, especially in coated buds. Since these buds contain endogenous albumin and lysosomal enzymes also, these data suggest that coated vesicles originating from TGR provide for a secretory route in Hep G2 cells and that this pathway is followed by the MPR system as well.Mannnose 6-phosphate receptors (MPR) 1 mediate the selective targeting of newly synthesized lysosomal enzymes to lysosomes. The receptors recognize mannose 6-phosphate residues which are added to the nascent enzyme molecules in, or in association with, the Golgi complex (1). After receptor-ligand binding, the complexes travel via an unknown pathway to the lysosomes. Before enzyme delivery, MPR and ligands uncouple in the acidic internal milieu of some prelysosomal compartment. Uncoupling permits free receptors to be re-used (2). The idea of recycling MPR stems mainly from studies on enzyme uptake by cells. These studies also indicate Abbreviations used in this paper. ASGP, asialoglycoprotein; CHO, Chinese hamster ovary; CURL, compartment of uncoupling receptors and ligands; GERL, region of smooth endoplasmic retieulum at the inner or trans-face of the Golgi stack; MPR, mannose 6-phosphate receptors; mvb's, multivesicular bodies; TGR, trans-Golgl reticulum.

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

“…At the trans-Golgi network (TGN)-endosomal interface, clathrin is implicated in sorting of cargo such as mannose 6-phosphate receptors (MPRs) (8), a pathway required for the lysosomal delivery of soluble acid hydrolases (9). CCP formation at the TGN involves the essential (10) heterotetrameric adaptor complex AP-1 (comprising the ␥, ␤1, 1, and 1 subunits), as well as the monomeric GGA (Golgi-localized, ␥-earcontaining, Arf-binding proteins) adaptors (11,12), in addition to a variety of accessory proteins including ␥-synergin, Eps15, Gadkin, and amphiphysin (13) among others (14).…”

Visualization of TGN to Endosome Trafficking through Fluorescently Labeled MPR and AP-1 in Living Cells

“…MPRs, their bound ligands as well as syntaxin 6, a SNARE protein involved in TGN-endosome trafficking, can be detected in vesicular profiles coated with clathrin and AP-1 assembly proteins (Geuze et al, 1985;Bock et al, 1997;Klumperman et al, 1998) located in close vicinity of the TGN. In polarized cells, AP-1B, the epithelial specific adaptor complex that differs from the ubiquitously expressed AP-1A by exchange of its 1A subunit by the closely related 1B, functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes (Folsch et al, 2001).…”

Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence.

“…The Golgi stack is composed of a variable number of flattened cisternae, in which an array of resident carbohydrate-modifying enzymes sequentially process the oligosaccharide chains of glycoproteins (Farquhar and Palade, 1981;Paulson and CoUey, 1989). Immediately apposed to the transmost aspect of the Golgi stack is the TGN (Roth et al, 1985;Geuze et al, 1985), a tubulo-reticular structure where proteins undergo further carbohydrate modifications and, in some cases, proteolytic processing (Duncan and Kornfeld, 1988). In addition to having a role in biosynthetic protein Dr. Peters is on sabbatical leave from the Department of Cell Biology, Medical School, University of Utrecht, The Netherlands.…”