Inhibition of p97-dependent protein degradation by Eeyarestatin I

Abstract

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-associated degradation involves substrate retrotranslocation from the ER lumen into the cytosol for degradation by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which hydrolyzes ATP to dislocate polyubiquitinated substrates into the cytosol. Polypeptides extracted by p97 are eventually transferred to the proteasome for destruction. In mammalian cells, ERAD can be blocked by a chemical inhibitor termed Eeyarestatin I, but the mechanism of EerI action is unclear. Here we report that EerI can associate with a p97 complex to inhibit ERAD. The interaction of EerI with the p97 complex appears to negatively influence a deubiquitinating process that is mediated by p97-associated deubiquitinating enzymes. We further show that ataxin-3, a p97-associated deubiquitinating enzyme previously implicated in ER-associated degradation, is among those affected. Interestingly, p97-associated deubiquitination is also involved in degradation of a soluble substrate. Our analyses establish a role for a novel deubiquitinating process in proteasome-dependent protein turnover.

FIGURE 1.

Accumulation of polyubiquitinated proteins in EerI-treated cells. A, structure of EerI. B, EerI treatment leads to an accumulation of polyubiquitinated HC in cytosol. A9 cells were treated with either Me₂SO (DMSO) or EerI (10 μm, 14 h), and permeabilized with the detergent digitonin. A portion of the cells was solubilized directly (T), whereas the rest of each sample was fractionated into a pellet (P) and a supernatant fraction (S). HC was immunoprecipitated (IP) with an anti-HA antibody under denaturing condition and analyzed by immunoblotting (IB). (HC+CHO, glycosylated HC; HC-CHO, deglycosylated HC; asterisk, a nonspecific protein cross-reacting with the ubiquitin antibody). C, as in B, except that A9 cells treated with either EerI or MG132 (10 μm) were analyzed. D, accumulation of polyubiquitinated TCRα in EerI-treated cells. Where as indicated, TCRα-GFP-expressing cells were treated with both EerI and MG132 (TCRα+CHO, glycosylated TCRα; TCRα-CHO, deglycosylated TCRα). A fraction of the extracts (WCE) was analyzed directly by immunoblotting. E, accumulation of polyubiquitinated proteins in EerI-treated cells. WCE of 293T cells treated with the indicated compounds were analyzed by immunoblotting.

FIGURE 2.

EerI inhibits PAD. A, scheme of the in vitro deubiquitination experiment. Ub-S, ubiquitinated substrates. B, EerI increases the level of p97-bound polyubiquitinated substrates. 293T cells treated with either Me₂SO (DMSO) or EerI (10 μm, 12 h) were lysed in a deoxyBigCHAP lysis buffer. A fraction of the lysate was analyzed directly by immunoblotting (IB)(WCE). The rest of the samples were subjected to immunoprecipitation with the indicated antibodies. Bottom panels are Coomassie Blue-stained gels. C, p97 and its associated proteins were immunoprecipitated from detergent extracts of DMSO-, MG132-, or EerI-treated cells, and incubated in vitro for the indicated time points. The graph shows the quantification of the experiment. D, as in C, except that ATP (5 mm) was included in the deubiquitinating reactions. Note that only 30% of the immunoprecipitated material for EerI-treated samples was loaded on the gel. The graphs show the quantification of two independent experiments (exp.).

FIGURE 3.

EerI inhibits atx3-dependent deubiquitination. A and B, atx3-dependent deubiquitination. A, FLAG-tagged atx3 and its associated proteins were immunoprecipitated from detergent extracts of atx3-expressing cells, and subjected to in vitro deubiquitination. Where indicated, the precipitated materials were incubated in the presence of ubiquitin aldehyde (Ub-A). B, ubiquitinated substrates bound to either FLAG-tagged wild-type atx3 (F-atx3) or to FLAG-tagged atx3 C14A (F-atx3 C14A) were analyzed by in vitro deubiquitination. C, EerI inhibits atx3-dependent deubiquitination. atx3 and its associated proteins immunoprecipitated from detergent extracts of Me₂SO (DMSO)- or EerI-treated cells (10 μm, 5 h) were incubated and analyzed as in A. The graph shows the quantification of the experiment. D, as in C, except that the atx3-substrate complex isolated from cells treated with MG132 was also included, and that the treatment time was 10 h.

FIGURE 4.

EerI does not disrupt the interaction between p97 and atx3. Detergent extracts of 293T cells treated with Me₂SO (DMSO) or EerI were subjected to immunoprecipitation with either control (IgG) or anti-p97 (αp97) antibodies. The immunoprecipitated material was immunoblotted (IB) with the indicated antibodies. Where indicated, a fraction of the extract (input, 15%) was directly analyzed by immunoblotting.

FIGURE 5.

EerI does not affect HAUSP-associated deubiquitination. 293T cells transfected with a plasmid expressing FLAG-tagged HAUSP together with HA-ubiquitin were treated as indicated. HAUSP and its associated substrates were immunoprecipitated from detergent extracts. HAUSP-mediated deubiquitination was analyzed. The graphs show the quantification of two independent experiments (exp.).

FIGURE 6.

EerI inhibits the degradation of a cytosolic p97 substrate. A, EerI inhibits the degradation of GFPμ. Astrocytoma cells stably expressing HA-tagged GFPμ were treated with the indicated chemicals (12 h). The steady state levels of GFPμ in WCE were analyzed by IB. Where indicated, GFPμ was first immunoprecipitated before immunoblotting. B, EerI does not affect the degradation of Ub-R-GFP. C, rate of GFPμ accumulation in EerI-treated cells correlates with that of an ERAD substrate. Stable cell lines expressing either GFPμ or TCRα-GFP were treated with EerI for the indicated time points. WCE were analyzed by IB.

FIGURE 7.

EerI associates with a p97 complex in cells. A, EerI emits fluorescence. a.u. artificial unit. B, association of EerI with p97. Endogenous p97 or Hsp90 was immunoprecipitated from extracts of EerI-treated 293T cells. The amount of EerI co-precipitated was determined by measuring the fluorescence associated with the precipitated material as described under “Experimental Procedures.” p97 or Hsp90 purified from untreated cells was used as blank. The immunoprecipitated proteins were also analyzed by IB with the indicated antibodies. Error bars show S.D. of five independent experiments. C, association of EerI with p97-atx3 complex. Endogenous p97 was immunoprecipitated (IP) with FLAG antibody from extract of EerI-treated cells that had been transfected with FLAG-atx3 (F-atx3). The amount of EerI present in the precipitated complex was determined as described under “Experimental Procedures.” As a control, cells transfected with an empty vector was used (–). The gel show the precipitated proteins determined by immunoblotting. Error bars show the S.D. of three independent experiments.

FIGURE 8.

EerI targets a p97 complex. A, EerI does not affect the association of p97 with Derlin-1 and VIMP. Endogenous proteins from extracts of 293T cells were analyzed by immunoprecipitation with the indicated antibodies followed by immunoblotting. B, EerI does not affect the association of p97 with Ufd1. C, EerI treatment alters a p97 complex. EerI-treated or -untreated astrocytoma cells were radiolabeled with [³⁵S]Met/Cys. Detergent extracts were subjected to immunoprecipitation with the indicated antibodies. Note that the short labeling time used did not allow the labeling of all p97 cofactors. Nonetheless, an unknown protein of 180 kDa (indicated by the arrow) was found in association with p97 only in untreated cells. D, interaction of the 180 kDa protein with p97 is exclusively on the membrane. Astrocytoma cells were treated with either Me₂SO (DMSO) or EerI, and permeabilized with the detergent digitonin. A portion of the cells was solubilized directly (T), whereas the rest of each sample was fractionated into a pellet (P) containing the ER membrane and a supernatant fraction (S) containing cytosol before subjected to solubilization and immunoprecipitation.