title: Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells.
abstract: We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.
authors.affiliation: European Molecular Biology Laboratory, Cell Biology Programme, Heidelberg, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Ludwig
authors.forename: G
authors.initials: G
authors.lastname: Griffiths
authors.forename: B
authors.initials: B
authors.lastname: Hoflack
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004705:Endocytosis; D006867:Hydrolases / Q000378:metabolism; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D007784:Lactoperoxidase / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008854:Microscopy, Electron; D010766:Phosphorylation; D051381:Rats; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism
chemical_list: D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D007784:Lactoperoxidase; D006867:Hydrolases