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.
Highlights
Substrate dislocation to the cytosol is a key step in the process of endoplasmic reticulum (ER) quality control
We propose that once in the cytosol, substrates must undergo a second round of ubiquitylation to engage the proteasome complex
N-Glycanase acts on glycoprotein substrates to remove attached N-linked glycan moieties, a reaction apparently dispensable for degradation [19], but experimentally quite informative because of the telltale reaction products that are diagnostic for dislocation
Summary
Substrate dislocation to the cytosol is a key step in the process of ER quality control. We show that addition of the purified EpsteinBarr virus deubiquitylase to semi-intact cells 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. We test the role of deubiquitylation in this model using an in vitro dislocation assay performed in semi-intact cells The configuration of this assay allows us to avoid dilution of cytoplasmic components, it enables manipulation, within certain limits, of the composition of the cytoplasmic environment through osmotic delivery of small molecules and proteins, including active and inactive versions of enzymes such as EBVDUB or trypsin, as well as ATP and GTP analogs. Because delivery of exogenous cytosol supports substrate dislocation in semi-intact cells, it should be possible to identify specific cytosolic components that are essential to this process
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