Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle.
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.
Hao
Mingming
M
Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
Lin
Sharron X
SX
Karylowski
Ola J
OJ
Wüstner
Daniel
D
McGraw
Timothy E
TE
Maxfield
Frederick R
FR
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*
D013261:Sterols; D014168:Transferrin; C010905:dehydroergosterol; D002784:Cholesterol; D004875:Ergosterol