Figure 7.

Effects of cholesterol and high-salt treatments on the GDI-mediated extraction of rab4 from endosomes in vitro. An enriched endosomal membrane fraction was prepared from HSFs or NPFs (see Materials and Methods), and the ability of GDI to extract rab4 from these membranes was quantified as in Figure 6. (A) Normal HSF endosomes were pretreated with 1 mM mβ-CD/cholesterol (increasing cholesterol levels approximately fourfold; see supplemental Figure 1, C) before GDI extraction. Note that GDI extraction of rab4 was selectively impaired and appeared similar to that seen for NPF endosomes (compare rab4 in Figure 6D for NPFs and Figure 7A for HSFs). (B) NP-C endosomal fractions were pretreated with 1 mM mβ-CD (decreasing cholesterol levels ∼2.5–3-fold; see supplemental Figure 1, C) before GDI extraction. Note that normal levels of GDI extraction of rab4 can be observed (compare rab4 in Figure 6D for NPFs and Figure 7B). (C) Endosome-enriched membrane pellets from HSFs and NPFs were briefly treated with 2 M KCl on ice, washed, and normalized for equal amounts of rab4 in treated (+KCl) and untreated (-KCl) membranes. The GDI extraction of rab4 was then quantified as in Figure 6, C and D. Note the extensive extraction of rab4 in KCl-treated NP-C membranes versus untreated membranes. (D) Quantitation of rab4 extraction from the indicated endosomal membrane fraction by GST-GDI was performed as in Figure 6D. Values are means ± SD from three independent experiments each in A, B, and D.