Abstract
Terminally misfolded glycoproteins are ejected from the endoplasmic reticulum (ER) to the cytosol and are destroyed by the ubiquitin proteasome system. A dominant negative version of the deubiquitylating enzyme Yod1 (Yod1C160S) causes accumulation of dislocation substrates in the ER. Failure to remove ubiquitin from the dislocation substrate might therefore stall the reaction at the exit site from the ER. We hypothesized that addition of a promiscuous deubiquitylase should overcome this blockade and restore dislocation. We monitored ER-to-cytosol transport of misfolded proteins in cells permeabilized at high cell density by perfringolysin O, a pore-forming cytolysin. This method allows ready access of otherwise impermeant reagents to the intracellular milieu with minimal dilution of cytoplasmic components. We show that addition of the purified Epstein-Barr virus deubiquitylase to semi-intact cells indeed initiates dislocation of a stalled substrate intermediate, resulting in stabilization of substrates in the cytosol. Our data provide new mechanistic insight in the dislocation reaction and support a model where failure to deubiquitylate an ER-resident protein occludes the dislocon and causes upstream misfolded intermediates to accumulate.