The sterol binding agent 2-hydroxypropyl-beta-cyclodextrin is shown to be a convenient and useful experimental tool to probe intracellular pathways of cholesterol transport. Biochemical and cytochemical studies reveal that cyclodextrin specifically removes plasma membrane cholesterol. Depletion of plasma membrane sphingomyelin greatly accelerated cyclodextrin-mediated cholesterol removal. Cholesterol arriving at the plasma membrane from lysosomes and the endoplasmic reticulum was also removed by cyclodextrin. Cellular cholesterol esterification linked to the mobilization of cholesterol from lysosomes was strongly attenuated by cyclodextrin, suggesting that the major portion of endocytosed cholesterol is delivered from lysosomes to the endoplasmic reticulum via the plasma membrane. Evidence for translocation of lysosomal cholesterol to the endoplasmic reticulum by a plasma membrane-independent pathway is provided by the finding that cyclodextrin loses its ability to suppress esterification when plasma membrane sphingomyelin is depleted. The Golgi apparatus appears to play an active role in directing the relocation of lysosomal cholesterol to the plasma membrane since brefeldin A also abrogated cyclodextrin-mediated suppression of cholesterol esterification. Using cyclodextrin we further show that attenuated esterification of lysosomal cholesterol in Niemann-Pick C cells reflects defective translocation of cholesterol to the plasma membrane that may be linked to abnormal Golgi trafficking.
Purified phosphatidylcholine exchange protein was used to exchange phosphatidylcholine between homogeneous single-walled phosphatidylcholine vesicles and human erythrocyte ghosts. When excess ghosts were present, it was found that only 70% of the vesicle phosphatidylcholine was available for exchange. This fraction corresponds closely to the amount of phosphatidycholine in the outer monolayer of these vesicles, indicating that only the outer surface of the vesicle is accessible to the exchange protein. Also, it was found that all phosphatidylcholine introduced into vesicles by the exchange protein was available for subsequent exchange. Using the exchange protein, asymmetrical vesicles were prepared in which the outer monolayer was either enriched or depleted in radioactive phosphatidylcholine as compared to the inner monolayer. Re-equilibration of the radioactivity between the two surfaces of the vesicle (flip-flop) could not be detected, even after 5 days at 37degrees. It is estimated that the half-time for flip-flop is in excess of 11 days at 37degrees. These results indicate that the properties of the exchange protein can be expolited to measure phosphatidylcholine flip-flop rates and possible phosphatidylcholine asymmetry in biological and model membranes, without altering the structure of the membrane.
The endoplasmic reticulum is the principal site of synthesis and initial incorporation of membrane lipids in eukaryotic cells; the enzymes of glycerolipid biosynthesis are exclusively located on its cytoplasmic surface. To maintain a phospholipid bilayer in this organelle, newly synthesized phospholipids must be translocated to the lumenal surface. Consistent with this are measurements indicating that movement of phospholipids across microsomal membranes is rapid, with a half-time less than 5 min (refs 3 and 4). Rapid movement of phospholipids has also been detected across the plasma membrane of Bacillus megaterium, another site of de novo lipid biosynthesis. The rapid transmembrane movement of phosphatidylcholine has not been detected, however, in vesicles prepared from microsomal lipids. These latter data suggest involvement in the endoplasmic reticulum of a phospholipid-translocating protein, as was first proposed by Bretscher who called it 'flippase'. Here we report reconstitution of a phospholipid flippase from rat liver microsomes into lipid vesicles.
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