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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.

Cholera toxin travels from the cell surface of affected mammalian cells to the endoplasmic reticulum (ER), where the A1 chain is released and retro-translocated across the ER membrane into the cytosol. We have tested whether, as in other cases, retro-translocation requires poly-ubiquitination. We show that an A1 chain mutant that lacks lysines and has a blocked N-terminus, and therefore cannot be ubiquitinated, remains active in vivo. The A1 chain is not degraded in the cytosol, as demonstrated by the fact that proteasome inhibitors do not stimulate its activity. When additional lysines are introduced into the A1 chain, moderate degradation by the proteasome is observed. The unfolded A1 chain rapidly refolds in vitro. These results show that poly-ubiquitination is not required for retro-translocation of all proteins across the ER membrane and indicate that the reason why the toxin escapes degradation in the cytosol may be both its paucity of lysines and its rapid refolding.