Retrograde transport of cholera toxin from the plasma membrane to the endoplasmic reticulum requires the trans-Golgi network but not the Golgi apparatus in Exo2-treated cells

Abstract

Cholera toxin (CT) follows a glycolipid-dependent entry pathway from the plasma membrane through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER) where it is retro-translocated into the cytosol to induce toxicity. Whether access to the Golgi apparatus is necessary for transport to the ER is not known. Exo2 is a small chemical that rapidly blocks anterograde traffic from the ER to the Golgi and selectively disrupts the Golgi apparatus but not the TGN. Here we use Exo2 to determine the role of the Golgi apparatus in CT trafficking. We find that under the condition of complete Golgi ablation by Exo2, CT reaches the TGN and moves efficiently into the ER without loss in toxicity. We propose that even in the absence of Exo2 the glycolipid pathway that carries the toxin from plasma membrane into the ER bypasses the Golgi apparatus entirely.

Figure 1

Exo2 selectively disrupts Golgi apparatus but not TGN. (A) Chemical structure of Exo2. (B) Exo2 blocks VSVG exit from ER. Time lapse of VSVG^ts045-GFP after temperature shift from 40 to 32°C. (C–E) Exo2 selectively disrupts Golgi apparatus. Cells treated with 50 μM of Exo2 or 5 μM of BFA were stained for KDEL receptor (C), clathrin adapter AP1 (D) and TGN protein TGN46 (E).

Figure 2

Exo2 inhibits Cl⁻ secretion in T84 cells. (A) Short-circuit current (I_sc, mean±s.d.) in polarized T84 cells treated with CT alone (open circles), Exo2 alone (open squares), CT with Exo2 (solid squares), and CT with BFA (solid triangles). Forskolin was added at 140 min (Fsk, arrows). (B) Exo2 acts downstream of cAMP. T84 cells were pretreated with vehicle alone DMSO (circles), BFA (triangles) or Exo2 (squares), and I_sc (arrow) was induced with forskolin (filled symbols) or 8-Br-cAMP (open symbols). Averages of duplicates are shown. (C,D) Maximal Cl⁻ secretion responses induced by CT (C) or anthrax EF (D) in the presence or absence of forskolin.

Figure 3

Exo2 collapses the Golgi apparatus in polarized T84 cells. (A) Confocal Z-series of T84 monolayers treated with Exo2 (50 μM) or BFA (5 μM) labelled for AP1, COPI coatomer and KDEL receptor. (B) Phosphorimage of metabolically labelled VSVG-GFP pulse (0 h) and acquisition of endoH resistance (r, upper band) in T84 monolayers treated with DMSO alone (upper panel), Exo2 (middle panel) or brefeldin A (lower panel).

Figure 4

Exo2 does not inhibit CT transport from TGN to ER. (A) Phosphorimage showing in duplicate the ³⁵S-sulphation (lower band) and N-glycosylation (upper band) of CTB-GS internalized for 3 h in T84 cells in the absence of vehicle (control), or treated with DMSO or Exo2. An immunoblot of the total cell lysate shows equal loading of CTB-GS (total). (B) Phosphorimage showing the ³⁵S-sulphation (lower band) and N-glycosylation (upper band) of CTB-GS internalized for 1 or 3 h in Vero cells treated with DMSO, Exo2, brefeldin A or cycloheximide (CHX).