title: Biosynthesis of the mannose 6-phosphate recognition marker in transport-impaired mouse lymphoma cells. Demonstration of a two-step phosphorylation.
abstract: The biosynthesis of the mannose 6-phosphate recognition marker has been studied in transport-impaired mouse lymphoma cells to determine the subcellular location of the processing enzymes and to characterize the biosynthetic intermediates. Cells were labeled with [2-3H]mannose and chased at a low temperature (15 or 20 degrees C) or at 37 degrees C in the presence of m-chlorocarbonylcyanide phenylhydrazone to disrupt transport of the pulse-labeled molecules within the secretory apparatus. Both treatments inhibited the migration of the pulse-labeled glycoproteins to the Golgi apparatus as measured by the production of complex-type asparagine-linked oligosaccharides. Despite this inhibition in protein transport, acid hydrolases were phosphorylated. Structural analysis of the phosphorylated oligosaccharides indicated that the transport-impaired cells produced a single species of phosphorylated high mannose oligosaccharide; essentially all of the molecules contain a single phosphodiester group that is restricted to the alpha 1,6 branch of the oligosaccharide. The results suggest that synthesis of mannose 6-phosphate-bearing high mannose oligosaccharides occurs in an ordered, compartmentalized posttranslational process. The initial phosphorylation of newly synthesized acid hydrolases occurs at a pre-Golgi site and results in the production of high mannose-type units that contain a single phosphodiester group. In a subsequent compartment, probably within the Golgi apparatus, the monophosphorylated units may be converted to diphosphorylated forms. Finally, at a site distal to the phosphorylation reactions the diesters are hydrolyzed to reveal the mannose 6-phosphate recognition marker.
authors.affiliation: Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
authors.forename: D A
authors.initials: DA
authors.lastname: Lazzarino
authors.forename: C A
authors.initials: CA
authors.lastname: Gabel
mesh_terms: D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology*; D002352:Carrier Proteins / Q000096:biosynthesis*; D002460:Cell Line; D006600:Hexosephosphates / Q000378:metabolism*; D006867:Hydrolases / Q000378:metabolism*; D008223:Lymphoma / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism*; D008360:Mannosephosphates / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009570:Nitriles / Q000494:pharmacology*; D009844:Oligosaccharides / Q000096:biosynthesis*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D017527:Receptor, IGF Type 2
chemical_list: D002352:Carrier Proteins; D006600:Hexosephosphates; D008360:Mannosephosphates; D009570:Nitriles; D009844:Oligosaccharides; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D006867:Hydrolases; D008358:Mannose