Lumenal labeling of rat hepatocyte endocytic compartments. Distribution of several acid hydrolases and membrane receptors.
We used a combination of subcellular fractionation and lactoperoxidase-mediated iodination to examine the polypeptide compositions of three hepatocyte endocytic compartments: early endosomes, late endosomes, and lysosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase which binds specifically to asialoglycoprotein receptors was perfused through isolated rat livers at 37 degrees C. Subcellular fractions enriched in various endocytic compartments were then isolated by differential and isopycnic centrifugation, and the lactoperoxidase moiety of the internalized conjugate was used to catalyze the iodination of lumenal-facing proteins. The 125I profiles of early and late endosomes were strikingly similar after gel electrophoresis. Using immunoprecipitation, we directly identified and compared the relative amounts of the Na+,K(+)-ATPase and several different acid hydrolases and membrane receptors in all three fractions. The asialoglycoprotein receptor and the low density lipoprotein related protein were approximately nine times more abundant in early endosomes than late endosomes, suggesting that they recycle from early endosomes. In addition, cathepsin D, but not cathepsin L, beta-glucuronidase, and lgp 120, was detected in early endosomes; however, all of these molecules were detected in lysosomes. Our findings provide strong evidence that early endosomes mature into late endosomes and that there is either selective delivery or selective retention of hydrolases at discrete points in the endocytic pathway.
Casciola-Rosen
L A
LA
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Renfrew
C A
CA
Hubbard
A L
AL
D000818:Animals; D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D019286:Endopeptidase K; D066246:ErbB Receptors / Q000378:metabolism; D006867:Hydrolases / Q000032:analysis*; D007070:Immunoglobulin A / Q000378:metabolism; D008099:Liver / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology; D008247:Lysosomes / Q000737:chemistry / Q000201:enzymology; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism; D015388:Organelles / Q000737:chemistry* / Q000201:enzymology; D009961:Orosomucoid / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism; D011961:Receptors, Fc; D011971:Receptors, Immunologic / Q000032:analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; C045281:IgA receptor; D007070:Immunoglobulin A; D008360:Mannosephosphates; D009961:Orosomucoid; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D011961:Receptors, Fc; D011971:Receptors, Immunologic; D066246:ErbB Receptors; D006867:Hydrolases; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D002402:Cathepsin D; D000254:Sodium-Potassium-Exchanging ATPase