Distinct Steps in Dislocation of Luminal Endoplasmic Reticulum-associated Degradation Substrates

Abstract

Dislocation of endoplasmic reticulum-associated degradation (ERAD) substrates from the endoplasmic reticulum (ER) lumen to cytosol is considered to occur in a single step that is tightly coupled to proteasomal degradation. Here we show that dislocation of luminal ERAD substrates occurs in two distinct consecutive steps. The first is passage across ER membrane to the ER cytosolic face, where substrates can accumulate as ubiquitin conjugates. In vivo, this step occurs despite proteasome inhibition but requires p97/Cdc48p because substrates remain entrapped in ER lumen and are prevented from ubiquitination in cdc48 yeast strain. The second dislocation step is the release of accumulated substrates to the cytosol. In vitro, this release requires active proteasome, consumes ATP, and relies on salt-removable ERbound components, among them the ER-bound p97 and ER-bound proteasome, which specifically interact with the cytosol-facing substrates. An additional role for Cdc48p subsequent to ubiquitination is revealed in the cdc48 strain at permissive temperature, consistent with our finding that p97 recognizes luminal ERAD substrates through multiubiquitin. BiP interacts exclusively with ERAD substrates, suggesting a role for this chaperone in ERAD. We propose a model that assigns the cytosolic face of the ER as a midpoint to which luminal ERAD substrates emerge and p97/Cdc48p and the proteasome are recruited. Although p97/Cdc48p plays a dual role in dislocation and is involved both in passage of the substrate across ER membrane and subsequent to its ubiquitination, the proteasome takes part in the release of the substrate from the ER face to the cytosol en route to degradation.The endoplasmic reticulum-associated degradation (ERAD) 1 is a quality control process that selectively eliminates malfolded proteins or unassembled subunits of oligomeric proteins in the secretory pathway (1, 2). ERAD substrates are dislocated from the endoplasmic reticulum (ER) back to the cytosol via the Sec61 complex (3-6). In the cytosol, ubiquitin is conjugated to the ERAD substrates that are degraded by the proteasome (7). The proteolytically active proteasome has been implicated in the dislocation of ERAD substrates by virtue of their scarcity in cytosol of proteasome-inhibited cells (8 -11). Although stabilization of proteins in the secretory pathway by proteasome inhibitors is generally accepted as an indication for ERAD, ubiquitination of such proteins is a direct evidence for the access of the substrate to the cytosol (7). In most cases, however, accumulation of the multiubiquitinated ERAD substrates to detectable levels requires inhibition of the proteasome. If the proteasome is indeed required for dislocation (8 -11), then ubiquitination of ERAD substrates would not be observed. This applies especially to luminal ERAD substrates, as membrane ERAD substrates inherently display cytosolic domains, which may be ubiquitinated irrespective of dislocation (11-15). Therefore, it is our view that luminal ERAD substrates, whose dislocation is an ...

Dislocation of HMG-CoA Reductase and Insig-1, Two Polytopic Endoplasmic Reticulum Proteins, En Route to Proteasomal Degradation

“…By fractionating permeabilized proteasomeinhibited cells, we demonstrate that endogenous HMGR and a tagged version of its membrane region, as well as Insig-1-Myc, are released to the cytosol as full-length intact polypeptides. These results complement reports on the dislocation of luminal ERAD substrates (Rodighiero et al, 2002;Elkabetz et al, 2004;Afshar et al, 2005) and of membrane proteins with a single TMs (Huppa and Ploegh, 1997;Tortorella et al, 1998;Ye et al, 2003). Together with recent in vitro evidence for dislocation of ubiquitinated polytopic Ste6*p and Hmg2p (Garza et al, 2009) from yeast microsomes, it seems that, among the possible models suggested for dislocation of polytopic membrane ERAD substrates , complete extraction to the cytosol of intact polypeptide chains is a general feature of the delivery to the proteasome of this class of proteins.…”

“…Not only is this process energetically expensive but also it challenges the cell to prevent these proteins from forming insoluble aggregates when the hydrophobic TMs are exposed to the hydrophilic environment of the cytosol. Indeed, specific AAA-ATPases, such as p97, and other molecular chaperones provide the driving force for dislocation and prevention of protein aggregation (Nishikawa et al, 2001;Ye et al, 2001;Elkabetz et al, 2004;Huyer et al, 2004;Bar-Nun, 2005;Buck et al, 2007;Lipson et al, 2008). The current study describes a system that should allow dissecting the steps in dislocation of polytopic ERAD substrates en route to proteasomal degradation and identification of cellular components that take part in this process.…”

Characterization of the Physiological Turnover of Native and Inactivated Cytochromes P450 3A in Cultured Rat Hepatocytes: A Role for the Cytosolic AAA ATPase p97?

“…In contrast, the autophagic/lysosomal inhibitors had minimal effect on either parameter. Indeed, parallel IB analyses of these 35 S-CYP3A immunoprecipitates with rabbit anti-Ub IgGs yielded the characteristic HMM ladders of polyubiquitylated CYP3A, which were dramatically enhanced in the presence of proteasomal, but not autophagic/lysosomal inhibitors, with marked accumulation observed at 6 h relative to the vehicle-treated cultures (Fig. 1D).…”

“…1A). Accordingly, 35 S-radiometric quantitation of these CYP3A immunoprecipitates revealed that of the initial (0 h) 35 S-CYP3A content, 73.3 ± 39.2% remained at 1h, falling to 38.3 ± 16.1% at 6 h (Fig. 1A).…”

“…1B). When these 35 S-CYP3A immunoprecipitates were subjected to SDS-PAGE/PhosphorImager analyses, a relative decrease of the 55 kDa 35 S-CYP3A species was clearly detected at ≈ 1 h, and this loss was further enhanced at 6 h. In parallel, an increase in high molecular mass (HMM) 35 S-CYP3A species was observed at 1h, subsiding thereafter at 6 h. These findings are consistent with CYP3A protein ubiquitylation before its proteolytic degradation (Fig. 1C).…”