SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins

Abstract

Membrane and secretory proteins that fail to pass quality control in the endoplasmic reticulum are discharged into the cytosol and degraded by the proteasome. Many of the mammalian components involved in this process remain to be identified. We performed a biochemical search for proteins that interact with SEL1L, a protein that is part of the mammalian HRD1 ligase complex and involved in substrate recognition. SEL1L is crucial for dislocation of Class I major histocompatibility complex heavy chains by the human cytomegalovirus US11 protein. We identified AUP1, UBXD8, UBC6e, and OS9 as functionally important components of this degradation complex in mammalian cells, as confirmed by mutagenesis and dominant negative versions of these proteins.

Fig. 1.

AUP1 and UBXD8 are found in the ER. HeLa cells were fixed and incubated with anti-PDI antibody and affinity-purified antibodies against AUP1 (Upper) or UBXD8 (Lower) for immunofluorescence analysis. (Right) Merged images. (Scale bars: 10 μm.)

Fig. 2.

OS9 is crucial for dislocation of the terminally misfolded glycoprotein, RI₃₃₂. (A) HeLa cells were transduced with either empty vector (pLHCX), wild-type OS9, OS9 R188A, OS9 E212D, or GFP-OS9, using the same virus preparation as in Fig. S4. The five cell lines were then transfected with a construct that specifies truncated ribophorin, RI₃₃₂, pulse-labeled 36 h after transfection for 20 min with ³⁵S, chased for indicated time points, and then lysed in 1% SDS and immunoprecipitated with antibody raised against the luminal portion of ribophorin. The eluates were separated on 10% SDS/PAGE and visualized by autoradiography. # indicates a faster migrating version of RI₃₃₂. (B) Quantitation of the amount of RI₃₃₂ remaining at the indicated time points.

Fig. 3.

UBC6e is crucial for US11-mediated dislocation. (A) US11-expressing cells were transduced with either empty vector (pLHCX), UBC6e WT, or UBC6e C91S mutant. The three cell lines were treated with ZL₃VS pulse-labeled for 10 min with ³⁵S, chased for indicated time points, and lysed in 1% SDS; the lysate was then immunoprecipitated with anti-HC. The eluates were separated on 12% SDS/PAGE and visualized on film. (B) Quantitation of the amount of glycosylated HC to total HC counts. (C) US2-expressing cells were transduced with the same virus preparation used in a. The experiment was performed as in A. (D) Quantitation of the amount of glycosylated Class I MHC HC to total HC counts.

Fig. 4.

AUP1 and UBXD8 are crucial for US11-mediated dislocation. (A) US11-expressing cells were transduced with either empty vector (pLHCX), AUP1-GFP, UBXD8-GFP, or GFP-OS9. The experiment was performed as in Fig. 3. (B) Quantitation of the amount of glycosylated Class I MHC HC to total HC counts. (C) US2-expressing cells were transduced with the same virus preparation used in a and a pulse–chase experiment was performed as for a. (D) Quantitation of the amount of glycosylated Class I MHC HC to total HC counts. (E) GFP-tag on UBXD8 hinders recruitment of p97. UBXD8-WT and UBXD8-GFP were expressed in 293T cells and immunoprecipitated with anti-UBXD8 antibodies from digitonin extracts. The eluates were separated on a 10% SDS/PAGE and immunoblotted with anti-p97 antibodies.

Fig. 5.

Schematic of US11-mediated dislocation. Depicted is the complex that US11 uses to dislocate class I MHC HCs. Domains are italicized. Fn II, fibronectin type II domain; Glc II, glucosidase II domain.