Lumenal labeling of rat hepatocyte early endosomes. Presence of multiple membrane receptors and the Na+,K(+)-ATPase.
We used lactoperoxidase-mediated iodination to investigate the lumenal polypeptide composition of rat hepatocyte endosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase that binds specifically to hepatocyte asialoglycoprotein receptors was perfused through isolated rat livers at 16 degrees C in the presence of mannan, resulting in the accumulation of ligand in early endosomes. Endosome containing low density vesicle fractions were subsequently isolated from sucrose gradients of microsomes, and the lactoperoxidase moiety was used to catalyze the iodination of lumenal-facing proteins. After gel electrophoresis, 125I-labeled early endosomes reproducibly showed a distinct 125I-polypeptide profile containing prominently labeled bands migrating at 43, 52, 58, 90, 110, 135, 230, and greater than 300 kDa. The asialoglycoprotein receptor (43-, 52-, and 58-kDa subunits) was by far the predominantly labeled protein even when iodinations were performed under conditions of receptor-ligand dissociation, and we conclude that it is the most abundant hepatocyte early endosomal protein. Furthermore, the iodination profile of the three asialoglycoprotein receptor subunits differed strikingly from their chemical amounts. Using immunoprecipitation, we directly identified the Na+,K(+)-ATPase; to our knowledge, this is the first biochemical evidence for the Na+,K(+)-ATPase in rat hepatocyte early endosomes. We also directly identified receptors for mannose 6-phosphate, epidermal growth factor, transferrin, and polymeric IgA in 125I-labeled early endosomes.
Casciola-Rosen
L A
LA
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Hubbard
A L
AL
D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism*; D001345:Autoradiography; D002462:Cell Membrane / Q000201:enzymology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007784:Lactoperoxidase / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D011233:Precipitin Tests; D051381:Rats; D011919:Rats, Inbred Strains; D011971:Receptors, Immunologic / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D009961:Orosomucoid; D011971:Receptors, Immunologic; C017030:asialoorosomucoid; D007784:Lactoperoxidase; D000254:Sodium-Potassium-Exchanging ATPase