Uptake and transport of mannosylated ligands by alveolar macrophages. Studies on ATP-dependent receptor-ligand dissociation.

T Wileman,R Boshans,P Stahl

doi:10.1016/s0021-9258(17)39620-5

Abstract

During endocytosis, mannosylated ligands enter vesicles which have a density intermediate between that of the plasma membrane and secondary lysosomes. Mannosylated ligands are transferred from these vesicles to lysosomes. A solubilization-precipitation assay was used to study the dissociation of mannosylated ligands from their receptor. In whole cells dissociation was rapid (t 1/2 (37 degrees C) = 8 min) and took place before delivery of the ligand to lysosomes. Receptor-ligand dissociation within membrane vesicles, washed free of cytosol, could be induced by addition of ATP and GTP but not ADP. Receptor-ligand dissociation caused by manipulating the pH of the vesicles suggested that the pH within endosomes was lowered to 5.5 by addition of ATP. Dissociation was blocked by proton ionophores and Zn2+, but was unaffected by inhibitors of the F1, Fo-ATPase or the Na+,K+-ATPase. Dissociation did not require Na+ or K+ and was blocked by anion transport inhibitors. Dissociation was slowed in the absence of permeant anions (Cl-). Receptor-ligand complexes within vesicles isolated as early as 2 min following ligand internalization responded to addition of ATP. The results suggest that receptor-ligand dissociation in endosomes requires ATP, possibly to power endosomal acidification via an ATP-dependent proton pump. Dissociation is enhanced in the presence of permeant anions, suggesting the involvement of an anion channel or carrier.

Highlights

Mannosylated ligandsare transferred from theseves- surface [4], and two lines of evidence have suggested that icles to lysosomes
Subcellular Fractionation-Percoll density gradient fractionation was used to study the uptakeandintracellular transport of '251-P-glucuronidaseby the mannose receptor of macrophages.Fig. lA shows the separation of marker enzymes for secondary lysosomes (@-hexosaminidase) and plasma membrane after centrifugation of a macrophage particulate fraction on a 1.07 g/mi Percoll gradient. @-Hexosaminidasewas localized within two peaks; the bulk of the enzyme was sedimented at thebottom of the gradient with a mean density of 1.08g/ml
The separation of the plasma membrane from secondary lysosomes allowed a study of the uptake and transport of 8

Summary

Introduction

Mannosylated ligandsare transferred from theseves- surface [4], and two lines of evidence have suggested that icles to lysosomes. Receptor-ligand dissociation caused by manipulating thepH of thevesicles suggested that the pH within endosomes was lowered to 5.5 by addition mannose receptor is pH-dependent with less avid binding at acidic pH [5], it followsthat intravesicular acidification would enhance receptor-ligand dissociation. The recycling of the mannose receptor is inhibited by amines andproton ionophores [5,6].These agents dissipate intravesicular pH gradients and block delivery of ligands to lysosomes. This leads to anaccumulation of receptor-ligand complexes within of ATP. We have developed a functional assay to show that this dissociation is an ATP-dependent process that can be perturbed by agents that dissipate pH gradients.

Methods

Results

Conclusion