The retrotranslocation protein Derlin-1 binds peptide:N-glycanase to the endoplasmic reticulum

Abstract

The deglycosylating enzyme, peptide:N-glycanase, acts on misfolded N-linked glycoproteins dislocated from the endoplasmic reticulum (ER) to the cytosol. Deglycosylation has been demonstrated to occur at the ER membrane and in the cytosol. However, the mechanism of PNGase association with the ER membrane was unclear, because PNGase lacked the necessary signal to facilitate its incorporation in the ER membrane, nor was it known to bind to an integral ER protein. Using HeLa cells, we have identified a membrane protein that associates with PNGase, thereby bringing it in close proximity to the ER and providing accessibility to dislocating glycoproteins. This protein, Derlin-1, has recently been shown to mediate retrotranslocation of misfolded glycoproteins. In this study we demonstrate that Derlin-1 interacts with the N-terminal domain of PNGase via its cytosolic C-terminus. Moreover, we find PNGase distributed in two populations; ER-associated and free in the cytosol, which suggests the deglycosylation process can proceed at either site depending on the glycoprotein substrate.

Figure 1.

Association of PNGase with Derlin-1 in vivo. (A) Topology of Derlin-1 containing four transmembrane domains with both the N- and C-termini in the cytosol. (B) Interaction of PNGase with Derlin-1 analyzed by coimmunoprecipitation. HeLa cells were cotransfected with pcDNA vector and Derlin-1-GFP (lane 1), pEGFP and PNGase-HA (lane 2), or PNGase-HA and Derlin-1-GFP (lane 3) and immunoprecipitated with monoclonal anti-HA antibody. Reverse immunoprecipitation was also performed with polyclonal anti-GFP antibody. Immune complexes obtained were resolved on SDS-PAGE and immunoblotted with antibodies as indicated. (C) Epitope tag has no effect on the interaction of PNGase with Derlin-1. HeLa cells were cotransfected with pcDNA vector and PNGase-GFP (lane 1), pEGFP and Derlin-1-Myc (lane 2), or PNGase-GFP and Derlin-1-Myc (lane 3) and immunoprecipitated with monoclonal anti-Myc antibody. Reverse immunoprecipitation was carried out with polyclonal anti-GFP antibody. Immune complexes obtained were resolved on SDS-PAGE and immunoblotted with antibodies as indicated. (D) Calnexin, an ER membrane protein, did not coprecipitate with PNGase. HeLa cells transfected with pcDNA vector alone (lane 1) and PNGase-HA (lane 2) were immunoprecipitated with monoclonal anti-HA antibody, resolved on SDS-PAGE, and immunoblotted with polyclonal anti-HA and polyclonal anti-calnexin antibodies as indicated.

Figure 2.

The cytosolic C-terminus of Derlin-1 interacts with PNGase. (A) Constructs of Derlin-1-GFP used in this study. (B) Coimmunoprecipitation of PNGase-HA with Derlin-1-GFP: HeLa cell lysate coexpressing pcDNA vector and Derlin-1-GFP (lane 1), pEGFP vector and PNGase-HA (lane 2), PNGase-HA and Derlin-1-GFP (lane 3), or PNGase-HA and Derlin-1-GFPΔ187–251 (lane 4) were immunoprecipitated with monoclonal anti-HA antibody (left panel) or reverse immunoprecipitation with polyclonal anti-GFP antibody (right panel). Immune complexes were resolved on SDS-PAGE, and immunoblotting was performed with antibodies as indicated.

Figure 3.

PNGase colocalizes with Derlin-1. HeLa cells were expressed with the plasmids as indicated, stained with the corresponding antibodies, and visualized by fluorescent microscopy. (A) Colocalization of PNGase-HA with Derlin-1: Derlin-1 staining (green), PNGase-HA staining (red), merge of PNGase-HA and Derlin-1 (yellow shows the colocalization of two proteins). (B) Colocalization of PNGase-HA with calnexin: calnexin staining (green), PNGase-HA staining (red), merge of PNGase-HA and calnexin (yellow shows the colocalization of two proteins). (C) Colocalization of Derlin-1 with calnexin: Derlin-1 staining (green), calnexin staining (red), merge of Derlin-1 and calnexin (yellow shows the colocalization of two proteins). (D) Colocalization of pEGFP vector alone with PNGase-HA: pEGFP staining (green), PNGase-HA staining (red), merge of pEGFP vector alone and PNGase-HA. DAPI (blue) shows nuclear staining.

Figure 4.

A small fraction of membrane-bound PNGase associates with Derlin-1. (A) HeLa cells coexpressing PNGase-HA and Derlin-1-GFP were lysed in 1% digitonin buffer, and the resulting cell lysate was subjected to Superdex 200 HR10/30 analytical size exclusion chromatography column preequilibrated with 1% digitonin buffer. Fractions obtained were subjected to coimmunoprecipitation with anti-HA (monoclonal) to detect interaction between PNGase and Derlin-1. The immunoprecipitates, bound on protein A/G agarose beads, were eluted in 2× SDS-PAGE sample buffer resolved on SDS-PAGE, and analyzed by immunoblotting with polyclonal anti-HA or monclonal anti-GFP. A minor fraction of PNGase coelutes with the bulk of Derlin-1 (fractions 15–20) followed by the elution of free PNGase (fractions 22–29). (B) Distribution of PNGase in HeLa cells. Subcellular fractionation was performed as described in Materials and Methods. Shown are HeLa cell lysate (lane 1), membrane free cytosolic fraction (lane 2), and ER membrane fraction (lane 3). Equal volumes of all the fractions were subjected to immunoprecipitation with monoclonal anti-HA (for PNGase-HA) and polyclonal anti-GFP (for Derlin-1-GFP) followed by SDS/PAGE and immunoblotting with antibodies as indicated. Anti-Calnexin antibody and anti-α-tubulin antibody were used as markers for ER and cytosol, respectively (top and bottom right panels, respectively). (C) PNGase enzyme activity in subcellular fractions. Activity assay for PNGase was performed using 10 μl of each fraction as described in Materials and Methods. Shown are pcDNA vector alone (lane 1), total cell lysate from HeLa cells (lane 2), membrane free cytosolic fraction (lane 3), ER membrane fraction (lane 4), and yeast PNGase mutant C191A (lane 5) used as a negative control.

Figure 5.

The N-terminus of PNGase interacts with Derlin-1. (A) Deletion constructs of PNGase used in this study. (B) HeLa cell lysates obtained from cells coexpressing pcDNA vector alone and PNGase-GFP (lane 1), pEGFP vector alone and Derlin-1 Myc (lane 2), Derlin-1-Myc and PNGase-GFP (lane 3), Derlin-1-Myc and ΔMCPNGase-GFP (lane 4), Derlin-1-Myc and ΔCPNGase-GFP (lane 5), Derlin-1-Myc and ΔNCPNGase-GFP (lane 6), Derlin-1-Myc and ΔNPNGase-GFP (lane 7), and Derlin-1-Myc and ΔNMPNGase-GFP (lane 8) were subjected to immunoprecipitation with monoclonal anti-Myc antibody. Reverse immunoprecipitation was performed using polyclonal anti-GFP antibody. Immune complexes bound on protein A/G agarose beads were eluted with 2× SDS-PAGE sample buffer resolved on SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies as indicated.

Figure 6.

The interaction of N-terminus of PNGase with the cytosolic C-terminus of Derlin-1 was confirmed by in vitro GST-binding assay (A) and gel filtration (B–D). (A) GST or GST-Derlin-1-C expressed in E. coli DH5α were purified and allowed to bind on GSH-agarose beads. E. coli BL21(DE3) cell lysate expressing full-length PNGase-(His)₆ and ΔMCPNGase-(His)₆ were used for in vitro GST-binding assay followed by SDS-PAGE and immunoblotting with anti-His and anti-GST antibodies. Shown are PNGase-(His)₆ in cell lysate (lane 1), GSH beads incubated with GST alone and PNGase-(His)₆ (lane 2), GSH beads carrying GST-Derlin-1-C and PNGase-(His)₆ (lane 3), cell lysate ΔMCPNGase-(His)₆ (lane 4), GSH beads incubated with GST alone and ΔMCPNGase-(His)₆ (lane 5), and GSH beads incubated with GST-Derlin-1-C and ΔMCPNGase-(His)₆ (lane 6). (B) Size exclusion chromatography (Superdex 200 HR 10/30) profile after mixing ΔMCPNGase-His and GST-Derlin-1-C in a 1:1 M ratio. The inset shows immunoblots of the peak fractions with anti-His and anti-GST antibodies, respectively. The presence of both proteins in a single peak indicates formation of a complex. (C) Elution profile of free GST-Derlin-1-C. Inset shows the immunoblot of peak fractions with anti-GST antibody. (D) Elution profile of free ΔMCPNGase-His. Inset shows the immunoblot of peak fractions with anti-His antibody.

Figure 7.

Model of PNGase mediated deglycosylation of misfolded glycoproteins retrotranslocated via channel formed by Derlin-1 (MHC class I HCs is used as a model substrate; there may be other glycoprotein substrates that utilize this pathway). As shown initially, a progressive series of protein-protein interactions occur. Derlin-1 interacts with the HCs and targets these to the cytosol for degradation by the proteasome. As shown, the N-terminus of PNGase interacts with the cytosolic C-terminus of Derlin-1 and consequently membrane-bound PNGase facilitates deglycosylation of the HCs in a coretrotranslocational manner, CHO indicates the carbohydrate chains. In this model we propose that the degradation of misfolded glycoprotein substrates occurs in close vicinity of the ER. On deglycosylation, HR23B acts as a receptor for deglycosylated substrates (Katiyar et al., 2004) and targets them to the proteasome for degradation by virtue of its interaction with the S₄ subunit. HR23B is known to be present close to the ER and its C-terminus has been shown to interact with PNGase (Suzuki et al., 2001; Katiyar et al., 2004). (Other Derlin-1 interacting partners p97 and VIMP [Ye et al., 2004] are not shown in this model.) A retrotranslocation channel formed by Sec61 complex is also shown in this model that may be involved in the translocation of a subset of misfolded protein substrates. Recently, the Sec61 channel has been demonstrated to be a membrane receptor for the proteasome (Kalies et al., 2005), suggesting that the degradation of misfolded proteins does indeed occur in close vicinity to the ER. Whether the retrotranslocation channel formed by Derlin-1 also directly interacts with the proteasome remains to be determined.