Phosphorylation of the cation-independent mannose 6-phosphate receptor is closely associated with its exit from the trans-Golgi network.
We have previously shown that two serine residues present in two conserved regions of the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) cytoplasmic domain are phosphorylated in vivo (residues 2421 and 2492 of the full length bovine CI-MPR precursor). In this study, we have used CHO cells to investigate the phosphorylation state of these two serines along the different steps of the CI-MPR exocytic and endocytic recycling pathways. Transport and phosphorylation of the CI-MPR in the biosynthetic pathway were examined using deoxymannojirimycin (dMM), a specific inhibitor of the cis-Golgi processing enzyme alpha-mannosidase I which leads to the accumulation of N-linked high mannose oligosaccharides on glycoproteins. Upon removal of dMM, normal processing to complex-type oligosaccharides (galactosylation and then sialylation) occurs on the newly synthesized glycoproteins, including the CI-MPR which could then be purified and analyzed on lectin affinity columns. Phosphorylation of the newly synthesized CI-MPR was concomitant with the sialylation of its oligosaccharides and appeared as a major albeit transient modification. Phosphorylation of the cell surface CI-MPR was examined during its endocytosis as well as its return to the Golgi using antibody tagging and exogalactosylation. The cell surface CI-MPR was not phosphorylated when it entered clathrin-coated pits or when it moved to the early and late endosomes. In contrast, the surface CI-MPR was phosphorylated when it had been resialylated upon its return to the trans-Golgi network. Subcellular fractionation experiments showed that the phosphorylated CI-MPR and the corresponding kinase were found in clathrin-coated vesicles. Collectively, these results indicate that phosphorylation of the two serines in the CI-MPR cytoplasmic domain is associated with a single step of transport of its recycling pathways and occurs when this receptor is in the trans-Golgi network and/or has left this compartment via clathrin-coated vesicles.
Méresse
S
S
European Molecular Biology Laboratory, Cell Biology Programme, Heidelberg, Germany.
Hoflack
B
B
D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002412:Cations; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D006224:Cricetinae; D004705:Endocytosis; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008360:Mannosephosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000378:metabolism*
D002412:Cations; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008360:Mannosephosphates; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D010768:Phosphoserine; C027693:mannose-6-phosphate