Detection of Membrane Cholesterol by Filipin in Isolated Rat Liver Coated

We investigated the cholesterol content of highly purified populations of coated vesicles from rat liver by biochemical quantitation and by cytochemical electron microscopy using the polyene antibiotic filipin . Failure of this reagent to elicit its typical response for a cholesterolcontaining membrane, i .e ., a characteristically corrugated or rippled appearance by thin section analysis, had led to the hypothesis (Montesano, R., A . Perrelet, P . Vassalli, and L . Orci, 1979, Proc. Natl . Acad . Sci . USA., 76:6391-6395) that cholesterol is specifically excluded from the plasma membrane domains associated with coated pit regions . The present electron microscopic results showed that although the response of coated vesicle membranes to filipin was also negative, uncoated vesicles whose clathrin coats had been removed in vitro exhibited a strong filipin-positive response . Quantitated biochemically, the cholesterol-to-phospholipid ratio of the coated vesicles was found to be indistinguishable from that of control preparations of plasma membranes isolated from rat liver . Taken together, the results indicate that the filipin-negative response of coated vesicles (and probably also that of coated pits) is due not to abnormally low cholesterol content, but rather to the stabilizing influence of their enveloping clathrin coats which inhibit the characteristic structural expression of the filipin-cholesterol complexes . Coated pits are specialized regions of the plasma membrane which play a central role in the pathway whereby eucaryotic cells internalize molecules by receptor-mediated endocytosis (1-8) . As observed by thin section electron microscopy, coated pits appear as invaginations underlined by the characteristic bristle coat of clathrin protein (9-13). In an attempt to elucidate the distinctive qualities ofcoated pit regions, Montesano et al . (14) examined the cholesterol content ofthese areas by freeze-fracture and thin-section electron microscopy. Using the polyene antibiotic filipin, a cytochemical marker that specifically binds to cholesterol and related 3-ß-hydroxysterols, they found no visible reaction to the probe in coated pit regions . This was in contrast to a positive response in surrounding tracts of uncoated membrane. On the basis of this observation, they concluded that coated pits are deficient in cholesterol, and as such might represent specialized domains of membrane which act as molecular filters in removing the sterol from internalizing membrane (15) . Their results agreed with earlier biochemical (16) as well as cytochemical studies (17) which suggested that depletion ofcholesterol in the coated pit region might be important in attaining the degree of fluidity required for membrane curvature and in creating a THE JOURNAL OF CELL BIOLOGY " VOLUME 99 JULY 1984 315-319 membrane environment conducive to receptor clustering (14, 18) . In a recent report, McGookey et al . (19) used ferritin-labeled probes to identify endocytic vesicles involved in cellular uptake oflow-density lipoprotein in cultured human fibroblasts. They found that although the membranes of coated pits and coated vesicles were largely insensitive to filipin, uncoated vesicles, derived from coated vesicles during endocytosis of the ferritin-conjugated ligand, exhibited the characteristic morphologic response of a filipin-sensitive membrane . Although they could not rule out the possibility that cholesterol moves rapidly into the endocytic vesicles subsequent to dissociation of the clathrin coat, they also considered that the negative response ofcoated pits and coated vesicles to filipin could well be due to causes other than the absence of cholesterol. Specifically, attention was drawn to the possibility that interaction between the clathrin coat and the underlying membrane somehow inhibits either the binding of filipin to cholesterol or inhibits the characteristic membrane changes associated with the formation of such complexes (20) . To ascertain whether coated membranes do, in fact, contain detectable amounts of cholesterol, we examined the effect of 315 on A ril 5, 2017 D ow nladed fom Published July 1, 1984