Lumenal Labeling of Rat Hepatocyte Early Endosomes

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 "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, '261-labeled early endosomes reproducibly showed a distinct l2'1-polypeptide profile containing prominently labeled bands migrating at 43,52,58,90, 110, 135, 230, and >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 receptorligand 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 6phosphate, epidermal growth factor, transferrin, and polymeric IgA in '261-labeled early endosomes.