Redistribution of mannose-6-phosphate receptors induced by tunicamycin and chloroquine.

Abstract

The distribution of Man-6-P receptors was determined by immunoperoxidase cytochemistry in Clone 9 hepatocytes cultured in the presence or absence of tunicamycin and chloroquine, agents that perturb lysosomal enzyme sorting and lead to their secretion . In control (untreated) cells, receptors were localized in cis Golgi cisternae, coated vesicles, and in endosomes or lysosomes . After tunicamycin treatment, receptors were found in coated vesicles lined up along the cis cisternae but were not detected in endosomes or lysosomes. After chloroquine treatment, receptors were localized in large vacuolated endosomes or lysosomes but were not usually detected in Golgi cisternae or in coated vesicles . These results demonstrate a redistribution of receptors along the normal Man-6-P-dependent sorting pathway after these treatments . In ligand-deficient (tunicamycintreated) cells, immunoreactive receptors accumulate at the presumptive sorting site in the cis Golgi and are depleted from endosomes and lysosomes. When the intralysosomal pH is increased (by chloroquine treatment) preventing ligand-receptor dissociation, receptors accumulate at the presumptive delivery site (lysosomes and endosomes) and are depleted from the cis Golgi region. The findings also suggest that (a) ligand binding triggers movement of the receptor to endosomes or lysosomes, and (b) ligand dissociation triggers their movement back to the cis Golgi region.Recently we localized the mannose-6-phosphate receptor for lysosomal enzymes in several secretory and absorptive cell types by immunocytochemistry in an attempt to define the Man-6-P-dependent transport route and to identify the site at which sorting of lysosomal enzymes and secretory products takes place (1) . We localized the receptor in cis Golgi cisternae, in coated vesicles, and in endosomes and lysosomes ofseveral cell types. We have assumed that this distribution delineates the Man-6-P-dependent pathway by which lysosomal enzymes are delivered to lysosomes, and that the organelles mentioned represent stations along this pathway which correspond to the sorting site, the carrier, and the delivery site, respectively. On the basis of these results we proposed that normally lysosomal enzymes bearing the recognition marker are removed from the secretory pathway in the cis Golgi for delivery to lysosomes, bypassing the trans Golgi cisternae .Our previous findings applied to the steady-state distribution of Man-6-P receptors in several epithelia (hepatocytes, exocrine pancreas, and epididymis) in which the polarity of the Golgi stacks (cis vs. trans) is clear. In this paper, we have extended our inquiry to cultured cells and have determined the distribution of the receptor both at steady state (untreated cells) and under conditions in which the traffic of lysosomal 320 enzymes is perturbed by tunicamycin (2, 3) or chloroquine (3-6) treatment . In cells incubated with these agents, sorting oflysosomal enzymes does not take place owing either to the production of defective ligand (2, 3) or to a d...

Upregulation of the Endosomal-Lysosomal Pathway in the Trembler-J Neuropathy

“…9). Chloroquine, a weak base, is thought to inhibit lysosomal enzyme activity by increasing the pH of the acidic vesicles leading to the accumulation of proteins that are destined for degradation (Brown et al, 1984). After a 16 hr chloroquine treatment, we found a notable accumulation of PMP22, P0, and MBP in Tr J /ϩ nerves (Fig.…”

TGN exit of the cation-independent mannose 6-phosphate receptor does not require acid hydrolase binding

“…13,14,16,17,19 In this paper we used quantitative immuno-EM to study the subcellular distribution of endogenous CI-MPR in the presence and absence of Man-6-P-containing acid hydrolases. Our data support the model that the CI-MPR is a constitutively cycling receptor that does not require ligand binding to exit the TGN.…”

“…This resulted in depletion of the CI-MPR from endosomes and accumulation in coated vesicles in the Golgi region, 19 suggesting that ligand binding is required to trigger CI-MPR exit from the TGN. Tunicamycin, however, is a general inhibitor that in addition to acid hydrolases affects many glycoproteins.…”

Mouse hepatitis virus strain A59 is released from opposite sides of different epithelial cell types.

“…specific secretion requires the presence of specific targeting information in a protein's structure (Brown et al, 1984 ;Kondor-Koch et al, GH 1985 ;Gottlieb et al, 1986 ;Caplan et al, 1987). Because MHV-A59 is released almost exclusively through the apical surface, virus particles most probably expose a signal(s) recognized by the MDCK sorting machinery and which directs them into vesicles destined for apical delivery.…”