Transferrin receptors and cation-independent mannose-6-phosphate receptors deliver their ligands to two distinct subpopulations of multivesicular endosomes.
The distribution of transferrin receptors (Tf-R) was determined in Clone 9 hepatocytes and compared to that of 215 kDa, cation-independent mannose-6-phosphate receptors (M6P-R) by double labeling. Cells were allowed to take up exogenous human transferrin (Tf) for 5 to 30 min, after which Tf, Tf-R, and M6P-R were localized by immunofluorescence using specific antibodies. All these proteins were found to be concentrated in the juxtanuclear or Golgi region. When Clone 9 cells were treated with NH4Cl to trap M6P-R in endosomes (Brown, W. J., J. Goodhouse, M. G. Farquhar: J. Cell Biol. 103, 1235-1247 (1986)), the distribution of the two receptors differed: Tf-R remained the same as in controls, but M6P-R were localized in large vacuolated endosomes. To carry out double labeling experiments at the electron microscope level, transferrin gold conjugates (Tf-Au) were prepared, and M6P-R were detected by immunoperoxidase labeling. Tf-Au binding to the cell surface was specific as it was reduced approximately 70 to 79% in the presence of excess native Tf. When Clone 9 cells were incubated with Tf-Au at 37 degrees C for 5 to 30 min, or binding of Tf-Au was carried out at 4 degrees C followed by warming to 37 degrees C, Tf-Au was found within a peripheral tubulovesicular network and within multivesicular endosomes that were not labeled with anti-M6P-R. Other multivesicular endosomes of similar size and morphology were heavily labeled for M6P-R but contained little or no Tf-Au. Tf-Au and M6P-R were also found in separate endosomes in cells treated with NH4Cl. Native Tf was localized in the same compartments as Tf-Au by immunoperoxidase labeling of both Clone 9 cells and mouse myeloma cells. We conclude that in Clone 9 hepatocytes, Tf/Tf-R internalized from the cell surface and M6P-R bearing newly synthesized lysosomal enzymes from the Golgi deliver their ligands to two different subpopulations of multivesicular endosomes. The endosomal subpopulation visited by Tf/Tf-R is known to correspond kinetically to early endosomes. The endosomal subpopulation heavily labeled for M6P-R presumably represent a later endosomal compartment which serves as the junction point where endocytosed ligands and newly synthesized lysosomal enzymes enroute to lysosomes meet.
Woods
J W
JW
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510-8002.
Goodhouse
J
J
Farquhar
M G
MG
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002999:Clone Cells; D004705:Endocytosis; D005455:Fluorescent Antibody Technique; D006600:Hexosephosphates / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D008099:Liver; D008247:Lysosomes / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure; D008360:Mannosephosphates / Q000378:metabolism*; D008854:Microscopy, Electron; D015388:Organelles / Q000032:analysis* / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000032:analysis / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism
D006600:Hexosephosphates; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014168:Transferrin; D000643:Ammonium Chloride