The A I chain of the cholera toxin (CT) undergoes retrotranslocation to the cytosol across the endoplasmic reticulum (ER) membrane by hijacking ER-associated degradation (ERAD). In the cytosol the CT A1 chain stimulates adenylyl cyclase. The VCPUfd1-Np14 complex mediates retrotranslocation of emerging ER proteins. While one group reported that VCP is required for CT retrotranslocation, another group concluded the opposite. We show that VCP is dispensable for CT retrotranslocation, however RNAi of either Ufd1 or Np14 induces an increase in adenylyl cyclase activity induced by CT. RNAi of VCP, Ufd1 or Np14 did not affect adenylyl cyclase activity induced by forskolin. These findings are coherent with our previous report showing that depletion of Ufd1-Np14 accelerates ERAD of reporter substrates. To integrate contradictory results we propose a new model, where Ufd1-Np14 is a negative regulator of retrotranslocation, delaying the retrotranslocation of ERAD substrates independently of its association with VCP. (c) 2007 Elsevier Inc. All rights reserved.