We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER)
to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTAΔ), follow ER-associated degradation
(ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated
in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical
polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase
retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the
Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the
classical ERAD-L pathway as well as an ongoing ER–Golgi transport. However, the dislocation pathways deviate
strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTAΔ, although the involvement of
individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber
itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the
proteasome RP, can discriminate between structural features of the same substrate