Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p - PubMed (original) (raw)

Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p

Pedro Carvalho et al. Cell. 2010.

Abstract

Misfolded, luminal endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol and degraded by the ubiquitin/proteasome system. This ERAD-L pathway requires a protein complex consisting of the ubiquitin ligase Hrd1p, which spans the ER membrane multiple times, and the membrane proteins Hrd3p, Usa1p, and Der1p. Here, we show that Hrd1p is the central membrane component in ERAD-L; its overexpression bypasses the need for the other components of the Hrd1p complex. Hrd1p function requires its oligomerization, which in wild-type cells is facilitated by Usa1p. Site-specific photocrosslinking indicates that, at early stages of retrotranslocation, Hrd1p interacts with a substrate segment close to the degradation signal. This interaction follows the delivery of substrate through other ERAD components, requires the presence of transmembrane segments of Hrd1p, and depends on both the ubiquitin ligase activity of Hrd1p and the function of the Cdc48p ATPase complex. Our results suggest a model for how Hrd1p promotes polypeptide movement through the ER membrane.

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Figures

Figure 1. Bypassing ERAD components by Hrd1p overexpression

(A) The degradation of the misfolded luminal ER protein CPY*-HA was followed after inhibition of protein synthesis by cycloheximide in wild type (wt) cells or in cells overexpressing Hrd1p under the GAL promoter in the presence of galactose. Where indicated, genes for ERAD components were deleted. The graph shows quantification of the data. (B) As in (A), but with overexpression of Der1p. The overexpression was confirmed by immunoblotting with specific antibodies (not shown). (C) As in (B), but with overexpression of Usa1p. (D) As in (B), but with overexpression of Hrd3p. See also Figure S1 and Figure S2.

Figure 2. Usa1p-mediated oligomerization of Hrd1p is required for ERAD-L

(A) Hrd1p-HA and Hrd1p-Myc were co-expressed under the endogenous promoter in either wild type (wt) or usaΔ cells. Detergent-solubilized membranes were treated with the bifunctional crosslinkers DSS or EGS, as indicated. Following quenching of the crosslinking reaction, immunoprecipitation with HA-antibodies was performed. Bound proteins were analyzed by SDS-PAGE followed by immunoblotting with Myc or Usa1p antibodies. The arrowhead indicates the position of a crosslinked species containing both Hrd1p and Usa1p. (B) Hrd1p-HA and Hrd1p-Myc were co-expressed in cells containing either wild type Usa1p or Usa1p mutants with the indicated deletions. Detergent-solubilized membranes were subjected to immunoprecipitation (IP) with HA-antibodies and bound proteins were analyzed by SDS-PAGE and immunoblotting with HA-, Myc-, or Usa1p- antibodies. H and U indicate the segments in Usa1p that are responsible for interaction with Hrd1p and Usa1p, respectively. (C) Usa1p-HA was co-expressed with FLAG-Usa1p or with the indicated FLAG-tagged deletion mutants of Usa1p. Detergent-solubilized membranes were subjected to immunoprecipitation (IP) with HA-antibodies and bound proteins were analyzed by SDS-PAGE and immunoblotting with HA- or FLAG- antibodies. Lanes 5 and 8 and the corresponding lanes 13 and 16 show the results with two independent clones of the same construct. (D) Hrd1p-HA and Hrd1p-Myc or Hrd1(C399S)-HA and Hrd1(C399S)-Myc were co-expressed in cells containing or lacking Usa1p. Detergent-solubilized membranes were subjected to immunoprecipitation (IP) with HA-antibodies and bound proteins were analyzed by SDS-PAGE and immunoblotting with HA- or Myc- antibodies. For cells lacking Usa1p, two independent clones co-expressing Hrd1p-HA and Hrd1p-Myc (Lanes 5 and 6 and the corresponding lanes 13 and 14) or Hrd1(C399S)-HA and Hrd1(C399S)-Myc (Lanes 7 and 8 and the corresponding lanes 15 and 16) are shown. (E) Kinetics of CPY*-HA degradation in cells expressing either wild type Usa1p or the indicated deletion mutants. The levels of CPY*-HA were determined by immunoblotting at different time points after cycloheximide addition (Figure S3). Shown are the means and standard deviations of three independent experiments. See also Figure S3.

Figure 3. Site-specific in vivo crosslinking of an ERAD-L substrate

(A) A shortened version of CPY* containing the last 180 amino acids, including the glycosylation site, was fused to dihydrofolate reductase (DHFR) and three HA tags (sCPY*-DHFR-HA). Single amber stop codons (TAG) were introduced at different sites of the coding sequence. The stop codon was suppressed in vivo by expression of a suppressor tRNA that is charged with the photoreactive amino acid analog benzoyl phenylalanine (BPA) by a modified amino acyl tRNA synthetase. UV irradiation leads to crosslinks with proteins in close proximity of the photoreactive probe. The position of the probe is defined relative to the glycosylation site (position 0; arrow head; corresponds to position 124 in sCPY*-DHFR-HA), with amino acid residues upstream and downstream given negative and positive numbers, respectively. (B) Photoreactive probes were placed at the indicated positions and the cells were irradiated with UV light, as indicated. Detergent-solubilized membranes were subjected to immunoprecipitation with HA antibodies, and bound proteins were analyzed by SDS-PAGE and immunoblotting with HA antibodies. See also Figure S4.

Figure 4. Site-specific in vivo crosslinking of an ERAD-L substrate to Hrd1p complex components

(A) Photoreactive probes were placed at the indicated positions of the substrate sCPY*-DHFR-HA. The constructs were expressed in cells that harbor under the endogenous promoter Hrd1p fused to 13 Myc tags (Hrd1-Myc). After immunoprecipitation with HA antibodies, the bound material was analyzed by SDS-PAGE and immunoblotting with Myc antibodies. (B) As in (A), but with a narrower range of positions of the photoreactive probes. (C) As in (A), but in cells that express under the endogenous promoter the luminal domain of Hrd3p (amino acids 1–767) fused to 13 Myc tags (Hrd3(1–767)-Myc). (D) As in (A), but in cells that express under the endogenous promoter Der1p fused to 13 Myc tags (Der1-Myc). (E) As in (A), but in cells that express under the endogenous promoter Usa1p fused to 13 Myc tags (Usa1-Myc). (F) As in (A), but in cells expressing Myc-tagged Hrd1 or mini-Hrd1p. As indicated in the diagram, mini-Hrd1p was generated by deletion of the last four transmembrane segments (TMs) of Hrd1p. Ring refers to the Ring finger domain essential for ubiquitin ligase activity. The intensity of the crosslinks to mini-Hrd1p was normalized to that of wild type Hrd1p. The numbers are the average of three experiments. See also Figure S5, Figure S6 and Table S1.

Figure 5. Effect of luminal ERAD events on substrate-Hrd1p crosslinking

(A) sCPY*-DHFR-HA or a mutant lacking the glycosylation site with photoreactive probes at the indicated positions were expressed in cells together with Hrd1-Myc. Following UV irradiation, detergent-solubilized membranes were subjected to immunoprecipitation with HA antibodies and bound proteins were analyzed by SDS-PAGE and immunoblotting with Myc antibodies. The intensity of the crosslinked band for the glycosylation mutant is given as a percentage of the intensity obtained with sCPY*-DHFR-HA. The numbers are the average of two to four experiments. (B) As in (A), but with sCPY*-DHFR-HA expressed in wild type (wt) cells or cells lacking the indicated ERAD components. The numbers give percentage of crosslinking intensity relative to wild type and are the average of two to four experiments. (C) As in (A), but with sCPY*-DHFR-HA expressed in cells harboring either wild type Hrd3p or the indicated Hrd3p deletion mutants. The numbers are the average of two experiments. (D) As in (A), but with sCPY*-DHFR-HA expressed in yos9Δusa1Δ cells harboring the indicated Usa1p deletion mutants. See also Figure S7.

Figure 6. Mutations of ERAD components on the cytosolic side affect substrate-Hrd1p crosslinking

(A) sCPY*-DHFR-HA with photoreactive probes at the indicated positions was expressed in cells together with Myc-tagged Hrd1 or a mutant defective in its ubiquitin ligase activity (C399S). Following UV irradiation, detergent-solubilized membranes were subjected to immunoprecipitation with HA antibodies, and bound proteins were analyzed by SDS-PAGE and immunoblotting with Myc antibodies. The numbers give percentage of crosslinking intensity relative to wild type and are the average of four experiments. (B) As in (A), with either wild type (wt) or usa1Δ cells. The numbers are the average of two experiments. (C) As in (A), with either wild type (wt) cells or cells bearing the indicated temperature-sensitive alleles of components of the Cdc48 ATPase complex analyzed after 1hr incubation at the restrictive temperature of 37°C. The numbers are the average of two experiments. See also Figure S7.

Figure 7. Organization of the Hrd1 complex and its interaction with substrate

(A) The scheme summarizes interactions between Hrd1p complex components, identified in the present study and elsewhere (for references, see text). For each protein, the N-terminus is indicated. Although not demonstrated, the C-terminal domain of Der1p (dotted) likely interacts with the C-terminal segment of Usa1p. The arrow indicates interaction between two Usa1p molecules, which facilitates oligomerization of the complex. (B) The scheme shows the ERAD-L substrate at an early stage of retro-translocation, as analyzed by photo-crosslinking.

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Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p - PubMed (original) (raw)