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Vol. 14, Issue 7, 2689-2705, July 2003

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

Lydia K. Nyasae, Ann L. Hubbard ^*, and Pamela L. Tuma 

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Submitted December 12, 2002; Revised March 3, 2003; Accepted March 13, 2003
Monitoring Editor: Jennifer Lippincott-Schwartz

We examined the role that lipid rafts play in regulating apical protein trafficking in polarized hepatic cells. Rafts are postulated to form in the trans-Golgi network where they recruit newly synthesized apical residents and mediate their direct transport to the apical plasma membrane. In hepatocytes, single transmembrane and glycolipid-anchored apical proteins take the "indirect" route. They are transported from the trans-Golgi to the basolateral plasma membrane where they are endocytosed and transcytosed to the apical surface. Do rafts sort hepatic apical proteins along this circuitous pathway? We took two approaches to answer this question. First, we determined the detergent solubility of selected apical proteins and where in the biosynthetic pathway insolubility was acquired. Second, we used pharmacological agents to deplete raft components and assessed their effects on basolateral-to-apical transcytosis. We found that cholesterol and glycosphingolipids are required for delivery from basolateral early endosomes to the subapical compartment. In contrast, fluid phase uptake and clathrin-mediated internalization of recycling receptors were only mildly impaired. Apical protein solubility did not correlate with raft depletion or impaired transcytosis, suggesting other factors contribute to apical protein insolubility. Examination of apical proteins in Fao cells also revealed that raft-dependent sorting does not require the polarized cell context.

Abbreviations used: APN, aminopeptidase N; ASGP-R, asialoglycoprotein receptor; DPP IV, dipeptidyl peptidase IV; FB1, fumonisin B1; GPI, glycophoshatidylinositol; LPDM, lipoprotein deficient medium; mCD, methyl--cyclodextrin; 5'NT, 5'nucleotidase; pIgA-R, polymeric IgA receptor; PM, plasma membrane; SAC, subapical compartment; SM, sphingomyelin; TLC; thin layer chromatography; Tf-R, transferrin receptor; TMD; transmembrane domain.

Present address: Department of Biology, The Catholic University of America, Washington, DC.

^* Corresponding author. E-mail address: alh{at}jhmi.edu.

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