Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane

Abstract

Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by retrotranslocation into the cytosol, a pathway hijacked by certain viruses to destroy MHC class I heavy chains. The translocation of polypeptides across the ER membrane requires their polyubiquitination and subsequent extraction from the membrane by the p97 ATPase [also called valosin-containing protein (VCP) or, in yeast, Cdc48]. In higher eukaryotes, p97 is bound to the ER membrane by a membrane protein complex containing Derlin-1 and VCP-interacting membrane protein (VIMP). How the ubiquitination machinery is recruited to the p97/Derlin/VIMP complex is unclear. Here, we report that p97 interacts directly with several ubiquitin ligases and facilitates their recruitment to Derlin-1. During retrotranslocation, a substrate first interacts with Derlin-1 before p97 and other factors join the complex. These data, together with the fact that Derlin-1 is a multispanning membrane protein forming homo-oligomers, support the idea that Derlin-1 is part of a retrotranslocation channel that is associated with both the polyubiquitination and p97-ATPase machineries.

Fig. 1.

Interaction of E3 ligases with components of the Derlin-1 complex. (a) Extracts from 293T cells, transfected with an empty vector (control) or with constructs encoding Myc-tagged wild-type Hrd1 (Hrd1 wt Myc), or Myc-tagged Hrd1 mutant containing a mutation in its ring finger domain (Hrd1 C291A Myc), were analyzed by immunoprecipitation (IP) with Myc antibodies, followed by immunoblotting (IB) with the indicated antibodies. Ten percent of the extracts were analyzed directly by immunoblotting (input). (b) A detergent extract of dog pancreatic microsomes was analyzed by immunoprecipitation (IP) with the indicated antibodies, followed by immunoblotting (IB) with various antibodies. The asterisks indicate proteins crossreacting with the antibodies. (c) As in a, except that cells expressed Hrd1-Myc, FLAG-tagged gp78 (gp78-FLAG), or both. The asterisk indicates residual FLAG-gp78 left after incomplete stripping of the FLAG immunoblot (Top). (d) As in a, except that cells expressed FLAG-tagged Parkin (FLAG-Parkin). IgG, immunoglobins detected by the secondary antibodies.

Fig. 2.

Colocalization of Hrd1 and components of the Derlin-1 complex. (a) HeLa cells expressing GFP-tagged Hrd1 (Hrd1-GFP) (a1, a4, and a7, green) were also stained with antibodies to p97, VIMP, or Sec61α (a2, a5, and a8, red). (a3, a6, and a9) Merged images, in which the nuclei were also stained with DAPI (in blue). Note that punctae are seen only in Hrd1-Myc-expressing cells (for comparison, see nonexpressing cells indicated by arrows). (b) HeLa cells expressing Hrd1-GFP on its own (b1–b3) or together with His-tagged p97 (b4–b6, His-p97) were visualized for GFP expression (in green) and stained with Derlin-1 antibodies (in red). (b3 and b6) Merged images.

Fig. 3.

Mapping interactions among components of the Derlin-1 complex. (a) A GST fusion protein containing the cytosolic domain of Hrd1 (GST-Hrd1c) was immobilized on glutathione beads and incubated with either purified His-tagged p97 (His-p97) or a His-tagged p97 mutant lacking its N domain (His-p97ΔN). Bound proteins were separated by SDS/PAGE and stained with Coomassie blue. GST was used as a control. (b)Asin a, except that a GST fusion to the cytosolic domain of gp78 (GST-gp78c) was used and binding to His-p97 was tested. (c) GST fusions to either p97 (GST-p97) or the cytosolic domain of VIMP (GST-VIMPc) were tested together with GST-Hrd1c and GST-gp78c for binding to the purified His-tagged C-terminal, cytosolic domain of Derlin-1 (His-Derlin-1c). The asterisk indicates a contaminated protein, copurified with His-Derlin-1c. (d) His-p97 or His-tagged p97 mutants, defective in either ATP-binding (His-p97 AA) or -hydrolysis (His-p97 QQ) were immobilized with p97-specific antibodies on protein A beads, and tested for binding of His-Derlin-1c. As a control, His-p97 was omitted from the reaction. (e) Scheme of protein–protein interactions within the Derlin-1 complex. The thin arrow indicates a weak interaction.

Fig. 4.

Assembly of the Derlin-1 complex. (a) US11-expressing astrocytoma cells were treated with a proteasome inhibitor and labeled with [³⁵S]methionine. A detergent extract of these cells was subjected to immunoprecipitation under native condition (1st IP) with specific antibodies, as indicated (Upper). A fraction of the precipitated material was analyzed by a second immunoprecipitation (re-IP) with antibodies to MHC class I heavy chains (HC) (Lower). A portion (10%) of the extract was directly precipitated (direct IP) with either US11 or heavy chain (HC) antibodies under denaturing conditions (lanes 1 and 2). The samples were analyzed by SDS/PAGE and autoradiography. The asterisks (Upper) designate several unknown proteins that were coprecipitated with the indicated antibodies. HC + CHO and HC - CHO indicate glycosylated and deglycosylated HC, respectively. (b) US11-expressing cells were labeled with [³⁵S]methionine, permeabilized, and incubated with cytosol containing a p97 mutant defective in ATP hydrolysis for 45 min before they were solubilized in detergent (45 min). An aliquot of cells was solubilized immediately after permeabilization (0 min). The detergent extracts were fractionated on 10–25% sucrose gradient. The amount of MHC class I heavy chains in each fraction was determined by immunoprecipitation and autoradiography, and was plotted (Upper). A portion of each fraction was analyzed by immunoblotting (IB) with Derlin-1 and p97 antibodies. (c) Detergent extracts of US11-expressing cells were subjected to immunoprecipitation with either control IgG or indicated antibodies (lanes 1–4). The supernatant from the Derlin-1 precipitation (1st) was subjected to a second (2nd) immunoprecipitation with Derlin-1 antibodies (lane 5). The resulting supernatant was analyzed by immunoprecipitation with either p97 (lane 6) or VIMP (lane 7) antibodies.

Fig. 5.

Derlin-1 forms homo-oligomer. A detergent extract of 293T cells, transfected with constructs encoding either GFP or a GFP-fusion to Derlin-1 (Derlin-1-GFP), was subjected to immunoprecipitation (IP) with GFP antibodies, followed by immunoblotting (IB) with the indicated antibodies. Ten percent of the extract was directly analyzed by immunoblotting (input).

Fig. 6.

A model for the assembly of the Derlin-1 complex during the retrotranslocation of MHC class I heavy chains. US11 initially targets glycosylated heavy chain (HC + CHO) to Derlin-1, resulting in a complex containing Derlin-1, US11, and HC plus CHO, which can be precipitated with antibodies to Derlin-1 (αDerlin-1). Substrate-bound Derlin-1 is then joined by a membrane complex containing VIMP, p97, and an E3 ligase (E3). The unknown ligase would be recruited to the complex in a similar way as Hrd1 and gp78. The final complex can be immunoprecipitated with antibodies to either VIMP or p97. A fraction of heavy chains (HC - CHO) associated with this complex has emerged into the cytosol because these lack carbohydrate chains, removed by a cytosolic glycanase.