A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function.
The determinants on the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) required for lysosomal enzyme sorting have been analyzed. Mouse L cells deficient in the mannose 6-phosphate/insulin-like growth factor-II receptor were transfected with normal bovine CD-MPR cDNA or cDNAs containing mutations in the 67-amino acid cytoplasmic tail and assayed for their ability to target the lysosomal enzyme cathepsin D to lysosomes. Cells expressing the wild-type bovine CD-MPR sorted 67 +/- 2% of newly synthesized cathepsin D compared with the base-line value of 47 +/- 1%. The presence of mannose 6-phosphate in the medium did not affect the efficiency of cathepsin D sorting, indicating that the routing of the ligand-receptor complex is completely intracellular. Mutant receptors with the carboxyl-terminal His-Leu-Leu-Pro-Met67 residues deleted or replaced with alanines sorted cathepsin D below the base-line value. A mutant receptor with the outermost Pro-Met residues replaced with alanines sorted cathepsin D better than the wild-type receptor, indicating that the essential residues for sorting are the His-Leu-Leu sequence. Disruption of a putative casein kinase II phosphorylation site at Ser57 had no detectable effect on sorting. The mutant receptor with the five-amino acid deletion was able to bind to a phosphopentamannose affinity column, proving that its ligand binding site was grossly intact. Resialylation experiments showed that this mutant receptor recycled from the cell surface to the Golgi at a rate similar to the normal CD-MPR, indicating that the defect in sorting is at the level of the Golgi.
Johnson
K F
KF
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Kornfeld
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002402:Cathepsin D / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D005690:Galactose / Q000378:metabolism; D007700:Kinetics; D007739:L Cells; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009439:Neuraminidase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D012794:Sialic Acids; D009439:Neuraminidase; D001616:beta-Galactosidase; D002402:Cathepsin D; D005690:Galactose