Figure 10.

Establishment of a cause-and-effect relationship between STxB association with DRMs and its transport from EE to the TGN. (A) A 15-min treatment at 37°C of SLO-permeabilized HeLa cells with the indicated doses of mβCD led to a dose-dependent extraction of free cellular cholesterol. The means of two to three experiments are shown. Nontreated SLO-permeabilized cells had 5 μg of cholesterol/10⁶ cells. (B) Cholesterol extraction displaces STxB from DRM fraction 2. Note that STxB association with fraction 2 is lower than in Figure 6 due to an additional incubation at 37°C for cholesterol extraction (see MATERIALS AND METHODS). The means of two to three experiments are shown. (C) Generic protocols used to reconstitute EE-to-TGN transport of STxB. Perm., permeabilization in intracellular transport buffer/dithiothreitol at 37°C (see MATERIALS AND METHODS). Detection: incubation of permeabilized cells with radioactive sulfate in the absence of cytosol. Protocol 1: standard protocol (see below, D). Protocol 2: Comparison of sulfation efficiencies on TGN-localized STxB (40 min transport at 37°C in intact cells) in SLO-permeabilized and mβCD- or mock-treated cells (for results, see insert; means of three experiments). (D) STxB transport from EE to the TGN on SLO-permeabilized cells is inhibited when DRMs are destabilized by 15-min extraction of cellular cholesterol with the indicated doses of mβCD. Transport efficiency in the presence of cytosol is put to 100%. The means (± SEM) of three to six experiments are shown. (E) ATP- and cytosol-dependent Tf recycling to the plasma membrane is not affected by cellular cholesterol extraction. The percentage of released Tf over total cell associated Tf was determined, and the means (± SEM) of 6–10 experiments are shown. (F) Cholesterol back-addition. In a variation of protocol 1 (C), the cells were extracted for 10 min at 37°C with 10 mM mβCD from permeabilization on, incubated for 10 min at 37°C with the indicated concentrations of cholesterol-saturated mβCD, and then shifted for 30 min to 37°C in the presence of cytosol and radioactive sulfate. Note that cholesterol back addition partially rescued transport to the TGN. (G) Quantification of cholesterol back-addition. The quantities of total free cellular cholesterol under the indicated conditions were determined. In F and G, a representative of two experiments is shown.