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Papers In Press, published online ahead of print March 1, 2004
J. Lipid Res., doi:10.1194/jlr.M300440-JLR200

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Journal of Lipid Research, Vol. 45, 427-437, March 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells

Daniel Wüstner¹, Mousumi Mondal, Amy Huang and Frederick R. Maxfield²

Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021

² To whom correspondence should be addressed. e-mail: frmaxfie{at}med.cornell.edu

We analyzed the intracellular transport of HDL and its associated free sterol in polarized human hepatoma HepG2 cells. Using pulse-chase protocols, we demonstrated that HDL labeled with Alexa 488 at the apolipoprotein (Alexa 488-HDL) was internalized by a scavenger receptor class B type I (SR-BI)-dependent process at the basolateral membrane and became enriched in a subapical/apical recycling compartment. Most Alexa 488-HDL was rapidly recycled to the basolateral cell surface and released from cells. Within 30 min of chase at 37°C, 3% of the initial cell-associated Alexa 488-HDL accumulated in the biliary canaliculus (BC) formed at the apical pole of polarized HepG2 cells. Even less Alexa 488-HDL was transported to late endosomes or lysosomes. The fluorescent cholesterol analog dehydroergosterol (DHE) incorporated into Alexa 488-HDL was delivered to the BC within a few minutes, independent of the labeled apolipoprotein. This transport did not require metabolic energy and could be blocked by antibodies against SR-BI. The fraction of cell-associated DHE transported to the BC was comparable when cells were incubated with either Alexa 488-HDL containing DHE or with DHE bound to methyl-ß-cyclodextrin.

We conclude that rapid, nonvesicular transport of sterol to the BC and efficient recycling of HDL particles underlies the selective sorting of sterol from HDLs in hepatocytes.

Abbreviations: Alexa 488-HDL, Alexa 488-labeled high density lipoprotein; apoA-I, apolipoprotein A-I; BC, biliary canaliculus; DHE, dehydroergosterol; DHE/Alexa 488-HDL, Alexa 488-labeled high density lipoprotein containing dehydroergosterol; DHE/MßCD, DHE loaded on methyl-ß-cyclodextrin; DiI-C18 dioctadecanoyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; ERC, endocytic recycling compartment; MßCD, methyl-ß-cyclodextrin; PC, phosphatidylcholine; PFA, paraformaldehyde; ROI, region of interest; SAC/ARC, subapical compartment/apical recycling compartment; SR-BI, scavenger receptor class B type I; Tf, transferrin

Supplementary key words hepatocytes • dehydroergosterol • bile • fluorescence microscopy

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