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© The Rockefeller University Press, 0021-9525/1998//115 $5.00
The Journal of Cell Biology, Volume 141, Number 1, , 1998 115-133

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

Gudrun Ihrke^*, Greg V. Martin^*, Michael R. Shanks^*, Michael Schrader, Trina A. Schroer, and Ann L. Hubbard^*

^* Department of Cell Biology and Anatomy, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205; and Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218

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.

Abbreviations used in this paper: 5'NT, 5'-nucleotidase; APN, aminopeptidase N; ASGP-R, asialoglycoprotein receptor; BC, canalicular-like space(s); DPPIV, dipeptidyl peptidase IV; GPI, glycosyl phosphatidyl inositol; HSFM, Hepes-buffered serum-free medium; IMF, indirect immunofluorescence; M6P-R, mannose-6-phosphate receptor; pIgA-R, polymeric IgA receptor; pAb, polyclonal antibody; PM, plasma membrane; r.t., room temperature; SAC, subapical compartment; Tf, transferrin; Tf-R, transferrin receptor.

Address all correspondence to Ann L. Hubbard, Department of Cell Biology and Anatomy, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. Tel.: (410) 955-2333. Fax: (410) 995-1013. E-mail: alh{at}welchlink.welch.jhu.edu

2. Portions of this work have been presented in abstract form: Ihrke, G., K. Finnegan, and A.L. Hubbard. 1993. Transcytosis of apical plasma membrane proteins in the hepatoma-derived WIF-B cell line. Mol. Biol. Cell. 4:97a; Ihrke, G., and A.L. Hubbard. 1995. Trafficking of plasma membrane proteins in polarized WIF-B hepatocytes. Eur. J. Cell Biol. (Suppl.):214; Shanks, M.R., H. Fujita, and A.L. Hubbard. 1996. Microinjection of antibodies to detect delivery of membrane proteins at the apical surface in WIF-B cells. Mol. Biol. Cell. 7:517a.

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