The 46-kDa mannose 6-phosphate receptor does not depend on endosomal acidification for delivery of hydrolases to lysosomes

Abstract

In mammalian cells, the mannose 6-phosphate receptor pathway accounts for the transport of most soluble acid hydrolases to lysosomes. It is believed that dissociation of mannose 6-phosphate receptors and their ligands is entirely driven by the acidic environment in endosomal compartments. Indeed, pH-perturbing substances such as ammonium chloride and monensin have been shown to inhibit lysosomal enzyme targeting in cells that express both known mannose 6-phosphate receptors. We now demonstrate that ammonium chloride and monensin exert modest effects on the intracellular retention of lysosomal hydrolases in murine cells that synthesize only the 46-kDa mannose 6-phosphate receptor. Neither ammonium chloride nor monensin induces changes to the subcellular localization of lysosomal hydrolases and the 46-kDa mannose 6-phosphate receptor in these cells. This suggests that endosomal dissociation of the receptor and its ligands still occurs in the presence of these agents. We conclude that the murine 46-kDa mannose 6-phosphate receptor has the capacity to deliver its cargo proteins to lysosomes even in the absence of endosomal acidification.

The mannose 6‐phosphate/insulin‐like growth factor II receptor restricts the tumourigenicity and invasiveness of squamous cell carcinoma cells

“…30 Selection of stably transfected cells was achieved by virtue of their ability to grow in the presence of 350 lg/ml hygromycin B (Invitrogen). Drug-resistant clones were isolated after 14 days and tested for M6P/IGF2R production by immunofluorescence analysis.…”

“…30 Briefly, cells grown on glass coverslips were fixed with paraformaldehyde and then incubated with the following antibodies diluted in phosphate-buffered saline containing 0.1% saponin: rabbit anti-bovine M6P/IGF2R (1:100), mouse anti-human M6P/IGF2R (2 lg/ml), rabbit anti-mouse MPR46 (1:100), rabbit anti-mouse cathepsin D (1:100), rat anti-mouse LAMP-1 (1:100), mouse anti-rat GM130 (2 lg/ml) and mouse anti-rat protein-disulfide isomerase (2 lg/ml). Bound primary antibodies were detected by incubation with suitable fluorescein-or Cy3-conjugated affinity-purified secondary antibodies (Jackson ImmunoResearch, West Grove, PA).…”

“…29,34 Densitometric analysis of immunoblots was done as described. 30 Immobilization and biotinylation of M6P/IGF2R ligands Hansenula holstii NRRL Y-2448 phosphomannan (a kind gift of Dr. Morey E. Slodki, US Department of Agriculture, Peoria, IL) was rehydrated in 100 ml of 1% KCl at 4°C for 16 hr. The pH of the solution was adjusted to pH 2.5 by addition of Dowex 50W-X8 (H 1 -form) prior to incubation at 100°C for 1 hr.…”

“…Total cellular membranes prepared as described 30 were resuspended in 100 ll binding buffer (0.15 M NaCl, 50 mM imidazole/ HCl pH 7.0, 0.02% NaN 3 ) containing proteinase inhibitors (5 lg/ ml E-64, 5 lg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride) and extracted with 1% (w/v) Triton X-100 for 30 min at 0°C. Aliquots of these membrane extracts corresponding to 150 lg total protein were diluted 10-fold with binding buffer and incubated with 40 ll settled phosphomannan-Sepharose beads on an endover-end mixer for 16 hr at 4°C.…”

“…Furthermore, SCC-VII cells lack mature lysosomes and store their acid hydrolases instead in compartments reminiscent of late endosomes. 10,29,30 In this study, we have generated SCC-VII lines stably reconstituted with human M6P/IGF2R to directly assess the impact of this receptor on the tumourigenicity and invasiveness of squamous cell carcinoma cells. …”