Acidification-dependent dissociation of endocytosed insulin precedes that of endocytosed proteins bearing the mannose 6-phosphate recognition marker.
A key step in the sorting of endocytosed ligands from their receptors is dissociation, which is triggered by the acidic pH of endosomes. To determine whether dissociation occurs synchronously for all ligands, we compared in Chinese hamster ovary cells the intracellular dissociation of insulin, which dissociates between pH 6.3 and 7.0, with that of lysosomal hydrolases bearing the mannose 6-phosphate recognition marker (Man-6-P proteins), which dissociate around pH 5.8. Chinese hamster ovary cells were pulsed for 2 min with 125I-insulin, acid-washed to remove surface binding, and chased. During a 40-min period, about 50% of the internalized 125I-insulin was released intact via a retrocytotic pathway. Retrocytosis was not inhibited by monensin, suggesting that the release was not dependent on acidic endosomes. The remaining insulin dissociated from its receptor in an acidification-sensitive manner and was eventually degraded. Dissociation was 70% complete within 5 min of internalization. When cells were similarly incubated with 125I-Man-6-P proteins, about 35% of the internalized radioactivity was released during a 1-h chase, reflecting proteolytic maturation of the Man-6-P proteins. Dissociation of Man-6-P proteins was acidification-dependent (i.e. inhibited by monensin), and was 50% complete after about 11 min. The results indicate that acidification-dependent dissociation of ligands does not occur in a single step and suggest that multiple endocytic compartments are involved in receptor/ligand sorting.
Borden
L A
LA
Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
Einstein
R
R
Gabel
C A
CA
Maxfield
F R
FR
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis; D005260:Female; D006600:Hexosephosphates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D008360:Mannosephosphates / Q000378:metabolism*; D008985:Monensin / Q000494:pharmacology; D010053:Ovary; D011865:Radioisotope Dilution Technique; D017527:Receptor, IGF Type 2; D011972:Receptor, Insulin / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D006600:Hexosephosphates; D007328:Insulin; D007457:Iodine Radioisotopes; D008024:Ligands; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D008985:Monensin; D011972:Receptor, Insulin