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

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.Polarity is a fundamental characteristic of most eukaryotic cells, either as a transient phenomenon (e.g., in a moving fibroblast) or a permanent feature (e.g., of an epithelial layer) (Drubin and Nelson, 1996). In epithelial cells, polarity is evident at many levels. At the cell surface, the basolateral and apical membrane domains face different environments (internal and external, respectively) and each membrane contains a distinct set of proteins and lipids (Simons and Fuller, 1985). Acquisition of the fully polarized epithelial phenotype requires assembly of tight and adhering junctions, which serve as barriers separating the apical and basolateral surfaces, and the selective delivery of plasma membrane (PM)¹ molecules and/or their retention at each surface (Rodriguez-Boulan and Powell, 1992; Simons et al., 1992; Wollner and Nelson, 1992).There is great variety among epithelial cells in the way specific PM proteins reach the same or different destinations. For example, kidney-derived MDCK cells sort most apical and basolateral membrane components in the TGN and then export this cargo directly to the “correct” surface (Matter and Mellman, 1994), although a variant line was recently found that delivers Na⁺,K⁺-ATPase to all PM domains randomly and then achieves a predominant basolateral distribution by selective retention (Hammerton et al., 1991; Mays et al., 1995). In other epithelial cells, apical PM proteins are first transported to the basolateral surface and then subsequently transcytosed to the apical domain, with sorting occurring in the endocytic pathway. The extent to which this more circuitous or “indirect” pathway to the apical surface is used depends on the specific protein and cell type (Rodriguez-Boulan and Zurzolo, 1993; Matter and Mellman, 1994). For delivery of apical membrane proteins, hepatocytes in vivo appear to use the indirect pathway exclusively (Bartles et al., 1987; Schell et al., 1992; Maurice et al., 1994), whereas cultured HepG2 cells reportedly deliver selected membrane lipids directly from the TGN to the apical PM (Zaal et al., 1994).The structural information directing membrane proteins through the transcytotic pathway has been elucidated only for the polymeric IgA receptor (pIgA-R). It is a sacrificial receptor whose 103-amino acid cytoplasmic tail contains multiple signals that direct the protein through the secretory pathway and into the transcytotic branch of the endocytic system. pIgA-R''s final destination is the apical membrane where an 80-kD proteolytic fragment of the receptor''s ectodomain is released into the apical milieu. An important difference between the pIgA-R and resident apical PM proteins studied so far is that the latter usually have short cytoplasmic tails with no apparent sorting signal (e.g., aminopeptiase N APN] and dipeptidyl peptidase IV DPPIV]), or are glycosyl phosphatidyl inositol (GPI)- anchored (e.g., 5′-nucleotidase 5′NT]). Positive sorting information is present elsewhere in these proteins, e.g., the glycolipid anchor of GPI-proteins (Lisanti and Rodriguez-Boulan, 1990) and the large ectodomains of APN and DPPIV (Vogel et al., 1992, 1995; Weisz et al., 1992), but finer resolution of such global signals has not yet been attained.Many studies have described the membrane compartments involved in the basolateral-to-apical transcytosis of soluble and/or membrane-bound cargo (e.g., Bomsel et al., 1989; Brändli et al., 1990; Hayakawa et al., 1990; van Deurs et al., 1990; van Genderen and van Meer, 1995). Although it is now clear that multiple compartments participate, the existence of stations or carriers that are unique to the transcytotic pathway is still an open question (e.g., Barroso and Sztul, 1994, versus Apodaca et al., 1994), as are the number and location(s) of the sorting site(s) for transcytotic cargo versus cargo destined for the recycling or lysosomal branches of the endocytic system (for reviews see Courtoy, 1993; Sandoval and Bakke, 1994; Gruenberg and Maxfield, 1995; Mostov and Cardone, 1995). The remarkable plasticity of the endocytic system as well as the possibility of real differences in the transport of soluble and membrane cargo may explain some of the apparent paradoxes. Early immuno-EM studies reported that in hepatocytes in vivo, the pIgA-R shares clathrin-coated entry sites with receptors that recycle between the PM and endosomal compartments (asialoglycoprotein receptor ASGP-R] and mannose-6-phosphate receptor M6P-R]), but is then segregated from them at the level of peripheral endosomes (called compartment for uncoupling of receptors and ligands) (Geuze et al., 1984). In contrast, the entry site(s) for resident apical proteins transiently present at the basolateral surface is still unknown. However, in liver in situ, newly synthesized DPPIV colocalizes with transcytosing pIgA-R in subapical tubulovesicular structures, suggesting that, at least in these cells, the last steps of transcytosis are common (Barr and Hubbard, 1993). Moreover, transcytotic membranes can be isolated that contain pIgA-R and newly synthesized DPPIV (Barr et al., 1995). Nevertheless, the extent to which different membrane protein classes with a common destination share a common pathway is still unclear.The newly developed WIF-B cell line is an ideal in vitro model for studying PM protein trafficking in polarized hepatocytes (Ihrke et al., 1993; Shanks et al., 1994). WIF-B cells grow in monolayers and acquire a polarized phenotype reminiscent of hepatocytes in vivo; that is, neighboring cells form bile canalicular-like spaces (BC). Each BC is completely sequestered from the surrounding medium as well as the substratum and apical PM proteins are highly concentrated in the BC membrane. Tight junctions prevent mixing of apical and basolateral PM proteins and block diffusion of large molecules such as antibodies from the culture medium into the BC (Ihrke et al., 1993).As a first step toward understanding the transcytotic pathway(s) in WIF-B cells and ultimately in liver, we define here the intracellular trafficking pathways taken by three different classes of membrane proteins that pass through the basolateral membrane: (a) apical PM proteins and pIgA-R; (b) basolaterally recycling receptors; and (c) proteins of the endosomal/lysosomal pathway that cycle through the PM. These proteins were tracked in living WIF-B cells by labeling with specific antibodies at the basolateral surface and determining the distributions of the antigen–antibody complexes at later times. Antibodies to a variety of apical PM proteins and the pIgA-R were specifically and efficiently transcytosed from the basolateral to the apical domain; all passed through a prominent subapical compartment before fusion with the apical PM. In contrast, antibodies to cycling membrane proteins, such as the ASGP-R, transferrin receptor (Tf-R), and M6P-R, and the lysosomal membrane protein lgp120, did not appear to pass through the subapical compartment, but rather were directly transported to the intracellular compartments that contained the highest concentrations of their antigens at steady state. However, antibodies to endolyn-78, another endosomal/lysosomal membrane protein (Croze et al., 1989), appeared transiently in the apical region of the cells before accumulating in lysosomes. Thus, the trafficking of endolyn-78 resembled to some degree the transcytotic route of apical PM proteins and pIgA-R.Our observations verify that transcytosis is a pathway for the delivery of apical PM proteins to the apical surface in WIF-B cells, as is seen in hepatocytes in vivo. Our findings suggest that two successive sorting compartments operate in WIF-B cells. Basolaterally endocytosed proteins pass first through peripheral endosomes, the compartment from which most ASGP-R and transferrin receptor (Tf-R) molecules recycle; from there lysosomal proteins such as lgp120 are directed towards lysosomes whereas transcytotic molecules are sorted out for transport to the apical pole. However, segregation of apical residents from at least one endosomal/lysosomal marker, endolyn-78, appears to occur after these proteins are delivered to an endomembrane compartment in the subapical region.²