Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation

Journal Article · Tue Sep 01 00:00:00 EDT 1987 · J. Cell Biol.; (United States)

DOI:https://doi.org/10.1083/jcb.105.3.1241· OSTI ID:5841709

Bartles, J R; Feracci, H M; Stieger, B; Hubbard, A L

We have used pulse-chase metabolic radiolabeling with L-(/sup 35/S)methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor (ASGP-R)) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates.

• Research Organization: Johns Hopkins University School of Medicine, Baltimore, MD
• OSTI ID: 5841709
• Journal Information: J. Cell Biol.; (United States), Vol. 105:3
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
METABOLISM
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
CELL MEMBRANES
COMPARATIVE EVALUATIONS
ELECTROPHORESIS
FRACTIONATION
IMMUNOASSAY
IN VIVO
LIVER CELLS
METHIONINE
RATS
SULFUR 35
TRACER TECHNIQUES
ULTRACENTRIFUGATION
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CENTRIFUGATION
DAYS LIVING RADIOISOTOPES
DRUGS
EVEN-ODD NUCLEI
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAMMALS
MEMBRANES
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RODENTS
SEPARATION PROCESSES
SOMATIC CELLS
SULFUR ISOTOPES
VERTEBRATES
550501* - Metabolism- Tracer Techniques