Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like domain-dependent manner

Abstract

Accumulation of aberrant proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response pathway that helps the cell to survive under these stress conditions. Herp is a mammalian ubiquitin domain protein, which is strongly induced by the unfolded protein response. It is involved in ER-associated protein degradation (ERAD) and interacts directly with the ubiquitin ligase Hrd1, which is found in high molecular mass complexes of the ER membrane. Here we present the first evidence that Herp regulates Hrd1-mediated ubiquitylation in a ubiquitin-like (UBL) domain-dependent manner. We found that upon exposure of cells to ER stress, elevation of Herp steady state levels is accompanied by an enhanced association of Herp with pre-existing Hrd1. Hrd1-associated Herp is rapidly degraded and substituted by de novo synthesized Herp, suggesting a continuous turnover of the protein at Hrd1 complexes. Further analysis revealed the presence of multiple Hrd1 copies in a single complex enabling binding of a variable number of Herp molecules. Efficient ubiquitylation of the Hrd1-specific ERAD substrate α1-antitrypsin null Hong Kong (NHK) required the presence of the Herp UBL domain, which was also necessary for NHK degradation. In summary, we propose that binding of Herp to Hrd1-containing ERAD complexes positively regulates the ubiquitylation activity of these complexes, thus permitting survival of the cell during ER stress.

FIGURE 1.

Dynamics of Hrd1-associated Herp. A, extracts of HeLa cells exposed to either 150 nm thapsigargin (tg) or 10 μg/ml tunicamycin (tu) for the indicated times were subjected to immunoprecipitation with preimmune serum (ctrl IP) or anti-serum specific for Hrd1 (Hrd1 IP) or Herp (Herp IP). Cell extracts and immunoprecipitated proteins were analyzed by Western blotting as indicated. GAPDH was used as a control to compare protein amounts of the extracts used for immunoprecipitation. An unspecific band detected with the Herp antiserum in the cell extracts is marked with an asterisk. B, HeLa cells exposed to 150 nm thapsigargin for 6 h or untreated were pulse-labeled with [³⁵S]methionine for 60 min. The chase was performed in the absence or presence of CHX for the indicated times. Cell extracts were subjected to immunoprecipitation (IP 1) as indicated (ctrl IP, Herp IP, and p97 IP). Precipitates were washed with radioimmune precipitation buffer to dissociate coprecipitated Hrd1 that was captured in a second immunoprecipitation using Hrd1-specific antiserum (IP 2 Hrd1). Samples were analyzed by SDS-PAGE followed by autoradiography. Unspecific bands are marked with an asterisk.

FIGURE 2.

Oligomerization of Hrd1. A, HeLa cells (ctrl) and Hrd1-HTB cells (clone 6 and clone 36) were lysed and subjected either to immunoprecipitation using Hrd1 antiserum (Hrd1 IP) or to streptavidin-agarose pull-down (strept. PD Hrd1-HTB). GAPDH was used as a control, demonstrating similar protein concentrations in the cell extracts. Precipitates were analyzed by Western blotting as indicated. A dash indicates the position of a 72-kDa protein marker, one asterisk indicates the position of WT-Herp, and two asterisks indicate the position of Herp-HTB. short exp., short exposure; long exp., long exposure. B, Hrd1-HTB cell extracts (clone 36) were subjected to glycerol gradient centrifugation. Fractions were incubated with streptavidin-agarose to precipitate Hrd1-HTB-containing complexes, which were analyzed by immunoblotting as indicated. For comparison, sedimentation of PA28 (∼200 kDa) and proteasomes (∼700–3000 kDa) is indicated by bars labeled accordingly. C, Hrd1-HTB cells (clone 36) were treated with 50 μg/ml CHX for the indicated periods of time. Cell extracts (extr.) were subjected to streptavidin-agarose pull-down (strept. PD). Precipitates were analyzed by immunoblotting as indicated. GAPDH was used as a loading control.

FIGURE 3.

The Herp UBL domain is required for the ubiquitylation of NHK. A, HeLa cells transfected with the indicated constructs were cultured for 48 h and treated with MG132 for 6 h prior to harvesting. HRD1-M-MYC encodes the RING domain mutant Hrd1C329S. Cell extracts were analyzed by immunoblotting with antibodies specific for α-tubulin, Herp, Myc, α1-antitrypsin (α1-AT), or GFP as indicated. As the His-ubiquitin-GFP fusion protein (His-Ub-GFP) is cleaved at the ubiquitin N terminus by endogenous ubiquitin-specific hydrolases, GFP serves as transfection control. Extracts were subjected to Ni-NTA pull-down (Ni-NTA PD) to capture His-tagged ubiquitin conjugates, which were analyzed by Western blotting using antibodies specific for ubiquitin and α1-antitrypsin (α1-AT). Bands marked by asterisks may reflect unspecific His-Ub-independent binding of NHK to the beads as they are not detected in cells that do not express the substrate (lane 1). Although also observed in other lanes, these bands appear to be pronounced in lanes 2 and 5 due to higher steady state levels of NHK. B, upon transfection with the indicated constructs, HeLa cells were grown for 24 h followed by treatment with MG132 for 6 h. Cell extracts were then subjected to immunoprecipitation with an α1-antitrypsin-specific antibody (α1-AT-IP). Extracts and immunoprecipitated proteins were analyzed by Western blotting using antibodies for GAPDH, peroxidase-coupled α1-antitrypsin (α1-AT), Herp, Myc, and peroxidase-coupled FK2 (ubiquitin). Apart from staining ubiquitylated NHK (ub-NHK), the ubiquitin antibody FK2 appears to cross-react with immunoglobulin heavy chains (Ig) from the α1-antitrypsin antibody used for the immunoprecipitation. shGFP, GFP-specific shRNA; shHERP, HERP-specific shRNA.

FIGURE 4.

Efficient degradation of NHK requires the UBL domain of Herp. HeLa cells were cotransfected with NHK and either GFP-specific shRNA (shGFP), HERP-specific shRNA (shHERP), HERP-specific shRNA, and a plasmid encoding WT-Herp (shHERP + HERP) or HERPshRNA and a plasmid encoding HerpΔUBL (shHERP + HERPΔUBL). Upon 48 h after transfection, the cells were exposed to 10 μg/ml tunicamycin or 10 μg/ml tunicamycin and 50 μg/ml CHX as indicated. To identify glycosylated NHK species, cell extracts were treated with 1000 units of PNGaseF (PNGase). GAPDH, Herp, and HerpΔUBL as well as glycosylated (NHK+CHO) and non-glycosylated (NHK-CHO) NHK were detected by immunoblotting. Bands of glycosylated NHK were further analyzed by densitometry, normalized to GAPDH, and plotted. Error bars represent S.D. (n = 3).