The ubiquitin‐dependent protein degradation system has recently been implicated in downregulation of signal transducing receptors. Growth hormone receptor (GHR) cDNA was transfected into Chinese hamster ovary cells, which exhibit a temperature‐sensitive defect in ubiquitin conjugation (CHO‐ts20), as well as into wild‐type cells (CHO‐E36). Upon binding of growth hormone (GH), two GHR polypeptides dimerize and initiate signal transduction. In CHO‐E36 and in CHO‐ts20 at the permissive temperature the GHR was ubiquitinated and degraded in a GH‐dependent fashion. However, at the non‐permissive temperature in CHO‐ts20 cells, neither GH‐dependent uptake nor degradation of the GHR was observed, while in CHO‐E36 cells both GHR uptake and degradation were accelerated. Incubation of CHO‐E36 cells with inhibitors of endosomal/lysosomal function (NH4Cl, bafilomycin A1) markedly reduced ligand‐induced GHR degradation. Our results indicate that a functional ubiquitin conjugating system is required for GH‐induced endocytosis and that degradation of both the exoplasmic and cytoplasmic portions of the GHR occurs within the endosomal/lysosomal compartment.

Abstract. The intracellular distributions of the cationindependent mannose 6-phosphate receptor (MPR) and a 120-kD lysosomal membrane glycoprotein (lgpl20) were studied in rat hepatoma cells. Using quantitative immunogold cytochemistry we found 10% of the cell's MPR located at the cell surface. In contrast, lgpl20 was not detectable at the plasma membrane. Intracellularly, MPR mainly occurred in the trans-Golgi reticulum (TGR) and endosomes, lgpl20, on the other hand, was confined to endosomes and lysosomes. MPR was present in both endosomal tubules and vacuoles, whereas lgpl20 was confined to the endosomal vacuoles. In cells incubated for 5-60 min with the endocytic tracer cationized ferritin, four categories of endocytic vacuoles could be discerned, i.e., vacuoles designated MPR÷/lgpl20 -, MPR÷/Igpl20 +, MPR-/ lgpl20 ÷, and vacuoles nonimmunolabeled for MPR and lgpl20. Tracer first reached MPR÷/lgpl20 -, then MPR+/Igpl20 ÷, and finally MPR-/IgpI20 ÷ vacuoles, which are assumed to represent lysosomes.To study the kinetics of appearance of endocytic tracers in MPR-and/or lgpl20-containing pools in greater detail, cells were allowed to endocytose horseradish peroxidase (HRP) for 5-90 min. The reduction in detectability of MPR and lgpl20 antigenicity on Western blots, due to treatment of cell homogenates with 3'3-diaminobenzidine, was followed in time. We found that HRP reached the entire accessible pool of MPR almost immediately after internalization of the tracer, while prolonged periods of time were required for HRP to maximally access lgpl20. The combined data suggest that MPR÷/Igpl20 + vacuoles are endocytic vacuoles, intermediate between MPR÷/lgpl20 -endosomes and MPR-/Igpl20 + lysosomes, and represent the site where MPR is sorted from lgpl20 destined for lysosomes. We propose that MPR is sorted from lgpl20 by selective lateral distribution of the receptor into the tubules of this compartment, resulting in the retention of lgpl20 in the vacuoles and the net transport of lgpl20 to lysosomes.

An affinity-purified rabbit antibody against rat liver mannose 6-phosphate receptor (MP-R) was prepared. The antibody was directed against a 215 kd-polypeptide and it recognized both ligand-occupied and free receptor . Anti-MP-R was used for immunofluorescence and immunoelectron microscopy of cryosections from rat liver. MP-R was demonstrated in all parenchymal liver cells, but not in endothelial lining cells. MP-R labeling was found at the entire plasma membrane, in coated pits and coated vesicles, in the compartment of uncoupling receptor and ligand, and in the Golgi complex . Lysosomes showed only scarce MP-R label .In double-labeling immunoelectron microscopy, MP-R co-localized with albumin in the Golgi cisternae and in secretory vesicles with lipoprotein particles. Cathepsin D was associated with MP-R in the Golgi cisternae . This finding indicates that MP-R/cathepsin D complexes traverse the Golgi complex on their way to the lysosomes . The possible involvement of CURL in lysosomal enzyme targeting is discussed .Studies on biosynthesis oflysosomal enzymes have established that these enzymes are synthesized in the rough endoplasmic reticulum where they are co-translationally directed from the ribosomes to the lumena ofthe endoplasmic reticulum (ER)' . The newly synthesized lysosomal enzymes are subjected to a series of modifications including glycosylation, phosphorylation, and other changes in the structure of the carbohydrate and protein moieties as they are transported from the ER to the Golgi complex and lysosomes (for recent review see references 1 and 2). Increasing evidence indicates that the enzymes are segregated from secretory proteins by membrane receptorsthat recognize mannose 6-phosphate residues, which are present uniquely on lysosomal enzymes . Biosynthesis of mannose 6-phosphate residues in the enzymes is catalyzed by N-acetylglucosamine-l-phosphotransferase and N-acetylglucosaminyl phosphodiesterase . These enzymes are associated with Golgi membrane fractions of higher or intermediate buoyant density, which probably correspond to the cis or intermediate cisternae of the Golgi complex (3, 4). These fractions are rich in a-mannosidase I (3, 4), (presumably 'Abbreviations used in this paper: CURL, compartment of uncoupling receptor and ligand; ER, endoplasmic reticulum ; GERL, Golgiassociated endoplasmic reticulum lysosomes; MP-R, mannose 6-phosphate receptors.

The transmissible gastroenteritis coronavirus (TGEV) infects the epithelial cells of the intestinal tract of pigs, resulting in a high mortality rate in piglets. This study shows the interaction of TGEV with a porcine epithelial cell line. To determine the site of viral entry, LLC-PK1 cells were grown on permeable filter supports and infected with TGEV from the apical or basolateral side. Initially after plating, the virus was found to enter the cells from both sides. During further development of cell polarity, however, the entry became restricted to the apical membrane. Viral entry could be blocked by a monoclonal antibody to the viral receptor aminopeptidase N. Confocal laser scanning microscopy showed that this receptor protein was present at both the apical and basolateral plasma membrane domains just after plating of the cells but that it became restricted to the apical plasma membrane during culture. To establish the site of viral release, the viral content of the apical and basolateral media of apically infected LLC-PK1 cells was measured by determining the amount of radioactively labelled viral proteins and infectious viral particles. We found that TGEV was preferentially released from the apical plasma membrane. This conclusion was confirmed by electron microscopy, which demonstrated that newly synthesized viral particles attached to the apical membrane. The results support the idea that the rapid lateral spread of TGEV infection over the intestinal epithelia occurs by the preferential release of virus from infected epithelial cells into the gut lumen followed by efficient infection of nearby cells through the apical domain.

Abstract. Trophoblast-like BeWo cells form wellpolarized epithelial monolayers, when cultured on permeable supports. Contrary to other polarized cell systems, in which the transferrin receptor is found predominantly on the basolateral cell surface, BeWo cells express the transferrin receptor at both apical and basolateral cell surfaces (Cerneus, D.P., and A. van der Ende. 1991. J. Cell Biol. 114: 1149-1158. In the present study we have addressed the question whether BeWo cells use a different sorting mechanism to target transferrin receptors to the cell surface, by examining the biosynthetic and transcytotic pathways of the transferrin receptor in BeWo cells. Using trypsin and antibodies to detect transferrin receptors at the cell surface of filter-grown BeWo cells, we show that at least 80 % of newly synthesized transferrin receptor follows a direct pathway to the basolateral surface, demonstrating that the transferrin receptor is efficiently intracellularly sorted. After surface arrival, pulselabeled transferrin receptor equilibrates between apical and basolateral cell surfaces, due to ongoing transcytotic transport in both directions. The subsequent redistribution takes over 120 min and results in a steady state distribution with 1.5-2.0 times more transferrin receptors at the basolateral surface than at the apical surface. By monitoring the fate of surfacebound '25I-transferrin, internalized either from the apical or basolateral surface transcytosis of the transferrin receptor was studied. About 15 % of t25I-transferrin is transcytosed in the basolateral to apical direction, whereas 25 % is transcytosed in the opposite direction, indicated that the fraction of receptors involved in transcytosis is roughly twofold higher for the apical receptor pool, as compared to the basolateral pool. Upon internalization, both apical and basolateral receptor pools become redistributed on both surfaces, resulting in a twofold higher number of transferrin receptors at the basolateral surface. Our results indicate that in BeWo Cells bidirectional transcytosis is the main factor in surface distribution of transferrin receptors on apical and basolateral surfaces, which may represent a cell type-specific, post-endocytic, sorting mechanism.