title: Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle.
abstract: We examined the intracellular transport of sterol in living cells using a naturally fluorescent cholesterol analog, dehydroergosterol (DHE), which has been shown to mimic many of the properties of cholesterol. By using DHE loaded on methyl-beta-cyclodextrin, we followed this cholesterol analog in pulse-chase studies. At steady state, DHE co-localizes extensively with transferrin (Tf), a marker for the endocytic recycling compartment (ERC), and redistributes with Tf in cells with altered ERC morphology. Expression of a dominant-negative mutation of an ERC-associated protein, mRme-1 (G429R), results in the slowing of both DHE and Tf receptor return to the cell surface. [3H]Cholesterol is found in the same fraction as 125I-Tf on sucrose density gradients, and this fraction can be specifically shifted to a higher density based on the presence of horseradish peroxidase-conjugated Tf in the same organelle. Whereas vesicular transport of Tf and efflux of DHE from the ERC are entirely blocked in energy-depleted cells, delivery of DHE to the ERC from the plasma membrane is only slightly affected. Biochemical studies performed using [3H]cholesterol show that the energy dependence of cholesterol transport to and from the ERC is similar to DHE transport. We propose that a large portion of intracellular cholesterol is localized in the ERC, and this pool might be important in maintaining cellular cholesterol homeostasis.
authors.affiliation: Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
authors.forename: Mingming
authors.initials: M
authors.lastname: Hao
authors.forename: Sharron X
authors.initials: SX
authors.lastname: Lin
authors.forename: Ola J
authors.initials: OJ
authors.lastname: Karylowski
authors.forename: Daniel
authors.initials: D
authors.lastname: Wüstner
authors.forename: Timothy E
authors.initials: TE
authors.lastname: McGraw
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Maxfield
mesh_terms: D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000378:metabolism; D013261:Sterols / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism*
chemical_list: D013261:Sterols; D014168:Transferrin; C010905:dehydroergosterol; D002784:Cholesterol; D004875:Ergosterol