Apical Plasma Membrane Proteins and Endolyn-78 Travel through a Subapical Compartment in Polarized WIF-B Hepatocytes

Abstract

We studied basolateral-to-apical transcytosis of three classes of apical plasma membrane (PM) proteins in polarized hepatic WIF-B cells and then compared it to the endocytic trafficking of basolaterally recycling membrane proteins. We used antibodies to label the basolateral cohort of proteins at the surface of living cells and then followed their trafficking at 37°C by indirect immunofluorescence. The apical PM proteins aminopeptidase N, 5′nucleotidase, and the polymeric IgA receptor were efficiently transcytosed. Delivery to the apical PM was confirmed by microinjection of secondary antibodies into the bile canalicular-like space and by EM studies. Before acquiring their apical steady-state distribution, the trafficked antibodies accumulated in a subapical compartment, which had a unique tubulovesicular appearance by EM. In contrast, antibodies to the receptors for asialoglycoproteins and mannose-6-phosphate or to the lysosomal membrane protein, lgp120, distributed to endosomes or lysosomes, respectively, without accumulating in the subapical area. However, the route taken by the endosomal/lysosomal protein endolyn-78 partially resembled the transcytotic pathway, since anti–endolyn-78 antibodies were found in a subapical compartment before delivery to lysosomes. Our results suggest that in WIF-B cells, transcytotic molecules pass through a subapical compartment that functions as a second sorting site for a subset of basolaterally endocytosed membrane proteins reaching this compartment.

Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

“…Detection of Kir6.2 and SUR in subcellular fractions of MIN6 cells Equal volumes from the gradient fractions were separated on 9% (w/v) polyacrylamide gels then blotted onto Immobilon-P transfer membrane (Millipore, Watford, UK) and probed with organelle-specific antibodies against mGPDH (mitochondria), phogrin (large dense-core insulin-containing vesicle [LDCV] membranes) [30,35], TGN38 (Golgi) [31], insulin receptor (IR, plasma membrane); LAMP-1 (lysosomes) [36]; SREBP precursor (endoplasmic reticulum [ER]) [32]; 14-3-3-β (cytosol) [37]. The protein and insulin contents in each fraction were determined using a Pierce BCA Protein Assay Kit (Rockford, IL, USA) and a Mercodia Ultrasensitive Mouse Insulin ELISA Kit (Uppsala, Sweden), respectively.…”

Cellular function and molecular structure of ecto-nucleotidases

“…This includes rapid lateral motility, cellular sorting to the apical membrane surface of polarized cells, clustering in lipid rafts and also transmembrane signaling [250,252]. Accordingly, eN is sorted to the apical membrane of hepatic cells [253,254].…”

Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells

“…Anti-5Јnucleotidase (5ЈNT) (monoclonal and affinity purified polyclonal), -CD59 (affinity-purified monoclonals), and -Tf-R (polyclonal) were kindly provided by J.P. Luzio (Cambridge University, Cambridge, United Kingdom), P. Morgan (University of Wales College of Medicine, Cardiff, United Kingdom), and M. Farquhar (University of California, San Diego, San Diego, CA), respectively. Antibodies against aminopeptidase N (APN), asialoglycoprotein receptor (ASGP-R), CE9, pIgA-R, dipeptidylpeptidase IV (DPP IV), and HA321 were prepared in the Hubbard laboratory and have been described previously Barr and Hubbard, 1993;Ihrke et al, 1993;Shanks et al, 1994).…”

“…Cells were processed for indirect immunofluorescence as described previously (Ihrke et al, 1993). Alexa 488-or 568-conjugated secondary antibodies were used at 3-5 g/ml.…”

“…WIF-B and Fao cells were grown in a humidified 7% CO 2 incubator at 37°C as described previously (Ihrke et al, 1993;Shanks et al, 1994). Briefly, cells were grown in F-12 medium (Coon's modification), pH 7.0, supplemented with 5% fetal bovine serum.…”