Figure 9.

EE-to-TGN transport is inhibited under cholesterol extraction conditions. (A) Biotinylated STxB mutant was bound to HeLa cells on ice (condition 4°C) or internalized at 19.5°C into EE (conditions 19.5°C, −mβCD, +mβCD). The cells were then either directly placed on ice (4 and 19.5°C), or incubated with (+mβCD) or without (−mβCD) 10 mM mβCD for 60 min at 19.5°C before passage on ice. After MESNA (+) or mock (−) treatment on ice, the cells were lysed and biotinylated STxB was detected by Western. (B) STxB-Sulf₂ was internalized and cholesterol was extracted as described in A. Cells were then used to determine the amount of free cellular cholesterol (control cells contained 7.93 ± 1.88 μg of cholesterol/10⁶ cellules) or were shifted to 37°C for 30 min in the presence of radioactive sulfate. Note that transport to the TGN (sulfation) was significantly inhibited by cholesterol extraction. The inset shows a typical result of the sulfation reactions in the −mβCD and the +mβCD conditions.