Human XTP3-B forms an endoplasmic reticulum quality control scaffold with the HRD1-SEL1L ubiquitin ligase complex and BiP

Abstract

The recognition of terminally misfolded proteins in the endoplasmic reticulum (ER) and the extraction of these proteins to the cytoplasm for proteasomal degradation are determined by a quality control mechanism in the ER. In yeast, Yos9p, an ER lectin containing a mannose 6-phosphate receptor homology (MRH) domain, enhances ER-associated degradation (ERAD) of glycoproteins. We show here that human XTP3-B (hXTP3-B), an ER lectin containing two MRH domains, has two transcriptional variants, and both isoforms retard ERAD of the human alpha(1)-antitrypsin variant null Hong Kong (NHK), a terminally misfolded glycoprotein. The hXTP3-B long isoform strongly inhibited ERAD of NHK-QQQ, which lacks all of the N-glycosylation sites of NHK, but the short transcriptional variant of hXTP3-B had almost no effect. Examination of complex formation by immunoprecipitation and by fractionation using sucrose density gradient centrifugation revealed that the hXTP3-B long isoform associates with the HRD1-SEL1L membrane-anchored ubiquitin ligase complex and BiP, forming a 27 S ER quality control scaffold complex. The hXTP3-B short isoform, however, is excluded from scaffold formation. Another MRH domain-containing ER lectin, hOS-9, is incorporated into this large complex, but gp78, another mammalian homolog of the yeast ubiquitin ligase Hrd1p, is not. Based on these results, we propose that this large ER quality control scaffold complex, containing ER lectins, a chaperone, and a ubiquitin ligase, provides a platform for the recognition and sorting of misfolded glycoproteins as well as nonglycosylated proteins prior to retrotranslocation into the cytoplasm for degradation.

FIGURE 1.

Intracellular localization of transfected hXTP3-B. A, domain organization of hXTP3-B-long and -short isoforms. B, fluorescence microscopy of 293 cells co-transfected with hXTP3-B-short-mRFP and NHK-GFP. After fixing cells with 4% paraformaldehyde at room temperature, mRFP and GFP signals were detected by confocal microscopy (Carl Zeiss). C, Endo H and PNGase F digestion of hXTP3-B and hOS-9. Cultures of 293 cells were transfected with HA-tagged hXTP3-B-long or -short or a FLAG-tagged hOS-9 v1, immunoprecipitated with the appropriate epitope tag antibody, and incubated at 37 °C with Endo H for 3 h or with PNGase F for 1.5 h. The thick arrows indicate the nonglycosylated forms of hXTP3-B, and the dotted arrows show the N-glycosylated forms. The arrowhead corresponds to N-glycosylated hOS-9, and the thin arrow shows nonglycosylated hOS-9. The asterisk indicates a band that binds to Protein G-Sepharose beads nonspecifically.

FIGURE 2.

Effect of hXTP3-B on ERAD. A, effect of hXTP3-B on the terminally misfolded glycoprotein NHK. NHK degradation in transfected 293 cells was examined by immunoprecipitation (IP) of cell lysates pulse-labeled with [³⁵S]methionine/cysteine. Immunoprecipitates were separated by 10% SDS-PAGE. The positions of the molecular weight standards are shown on the left. Quantitative analysis by phosphorimaging is shown on the right; error bars indicate S.E. (n = 3). The C terminus of hXTP3-B was tagged with mRFP. ER-mCherry, which expresses mCherry in the ER, was transfected as a control. B, effect of hXTP3-B on nonglycosylated NHK-QQQ degradation. NHK-QQQ degradation was examined as in A. C, secretion and intracellular transport of wild-typeα₁-antitrypsin. Quantitative analysis of a typical experiment is shown on the right. Disappearance of the ER form (upper graph), detection of the Golgi form (in cell lysate fractions, gray arrow in the left panel), and secretion into the medium (lower graph) are shown.

FIGURE 3.

HRD1-SEL1L complex formation. A, HRD1-SEL1L complex formation in 3% digitonin lysis buffer. Cultures of 293 cells were transfected with mock, SEL1L, and/or HRD1-Myc/gp78-Myc. After metabolic labeling for 3 h, cell lysates were immunoprecipitated with the indicated specific antibodies. The asterisk indicates a band nonspecifically detected by Protein G-Sepharose beads. non-imm, nonimmune rabbit serum. B, Endo H and PNGase F digestion of SEL1L and HRD1-Myc. N-Glycosidase digestion was performed as described in the legend to Fig. 1B. The results of two independent experiments are shown in the upper (lanes 1–12) and lower (lanes 13–16) panels.^*, as in A. C, identification of endogenous SEL1L, BiP, and p97/VCP by immunoprecipitation. Cells transfected with the indicated plasmids were labeled with [³⁵S]methionine/cysteine for 3 h and extracted with 3% digitonin. The electrophoretic mobilities of proteins immunoprecipitated with the indicated antibodies (IP) are compared.

FIGURE 4.

Association of hXTP3-B with the membrane-anchored ubiquitin ligase complex. A, co-immunoprecipitation of hXTP3-B-long and -short with SEL1L. Cells mock-transfected or transfected with HA-tagged hXTP3-B or mouse EDEM1 (mEDEM1) were metabolically labeled for 16 h prior to extraction with 3% digitonin. non-imm, nonimmune rabbit serum. B, comparison of complex formation by hXTP3-B-long and HRD1 in lysis buffer containing 3% digitonin or 1% Nonidet P-40. Cells were labeled for 16 h.

FIGURE 5.

Complex formation by hXTP3-B and HRD1. A, immunoblot of 293 cells co-transfected with hXTP3-B-long-HA and HRD1-Myc/gp78-Myc. Cells were solubilized with 3% digitonin, and immunoprecipitates of hXTP3-B (HA-tagged) were separated and blotted with anti-c-Myc or anti-HA antibodies (lanes 4–6). Aliquots of the lysates (10% of the total volume) used for immunoprecipitation (IP) were loaded as input controls (lanes 1–3). Partially degraded fragments of HRD1-Myc were detected (lane 2), but only full-length HRD1-Myc co-immunoprecipitated with hXTP3-B (lane 5). B, indirect association of hXTP3-B and HRD1. Cells transfected with FLAG-tagged hXTP3-B and HRD1-Myc were extracted in either 3% digitonin (left) or 1% Nonidet P-40 (right) and subjected to immunoprecipitation followed by Western blotting.

FIGURE 6.

Fractionation of hXTP3-B and mouse EDEM1 (mEDEM1) by sucrose density gradient centrifugation. Cells transfected with hXTP3-B or mouse EDEM1 were extracted with 3% digitonin and fractionated using a 10–40% sucrose density gradient. Fractions were Western blotted with anti-FLAG (for hXTP3-B-short and -long) or anti-HA (for mouse EDEM1) antibodies to determine the sedimentation profiles of hXTP3-B-short and -long and mouse EDEM1. The brackets indicate the two separate regions of the gradient where hXTP3-B-long was detected. Fractions containing high molecular weight calibration proteins are indicated at the bottom by their estimated sedimentation coefficients (arrows). The positions of molecular weight standards for SDS-PAGE are shown on the left.

FIGURE 7.

High molecular weight complex formation by hXTP3-B-long. A, co-fractionation of endogenous hOS-9 and SEL1L with hXTP3-B-long. Lysates of cells transfected with hXTP3-B with or without HRD1-Myc/gp78-Myc were fractionated by sucrose density gradient centrifugation as in Fig. 6 and were subjected to immunoblotting with anti-FLAG to detect hXTP3-B-long or with antibodies against endogenous proteins. The position of SEL1L is indicated by the arrowheads, and the two hOS-9 variants are indicated by arrows. The high molecular weight fractions containing hXTP3-B-long are indicated by the brackets. The asterisks denote nonspecific signals detected by the anti-OS-9 antibody. B, distribution of HRD1 and gp78. Fractions were probed with antibodies against the epitope tags (FLAG for hXTP3-B and c-Myc for HRD1 and gp78) or endogenous proteins (OS-9, SEL1L, and p97/VCP). The arrows and arrowheads are as in A. gp78-Myc is indicated by the gray arrowhead.

FIGURE 8.

Co-immunoprecipitation of hOS-9 with SEL1L and BiP. A, domain organization of hOS-9 variants 1 and 2. Variants lacking the 15-amino acid region indicated by the arrow in variants 1 and 2 correspond to variants 3 and 4, respectively. B, complex formation by hOS-9 and SEL1L. Cells were transfected with FLAG-tagged hOS-9 v1 or v2, labeled for 3 h, and extracted with 1% Nonidet P-40 (left) or 3% digitonin (right). The extracts were analyzed by immunoprecipitation (IP) with specific antibodies. non-imm, nonimmune rabbit IgG. The asterisk indicates a nonspecific signal detected by Protein G-Sepharose beads.

FIGURE 9.

Physical interaction between SEL1L, HRD1, and hXTP3-B. A, co-immunoprecipitation of SEL1L, HRD1, and hXTP3-B-long from fractions separated by sucrose density gradient centrifugation. Lysates of cells transfected with mock or hXTP3B-long-FLAG + HRD1-Myc were separated by 10–40% sucrose density gradient centrifugation as in Fig. 6, and fractions containing the 27 S high molecular weight complex (fractions 14–17) were pooled and subjected to immunoprecipitation followed by Western blot analysis. Input control lanes (lanes 1 and 2) contained aliquots (10% of the total volume) of the pooled fractions. Since hXTP3-B-long migrates close to the immunoglobulin heavy chains used for immunoprecipitation, short and long exposures of the FLAG blot are shown to allow visualization of FLAG-tagged proteins. B, effect of XTP3-B RNAi on complex formation. Cell cultures were incubated with the indicated siRNA (30 nm) and after 24 h, HRD1-Myc was transfected. After 48 h of RNAi treatment, cells were metabolically labeled for 3 h and then extracted with 3% digitonin. Complex formation was analyzed by immunoprecipitation (IP) with specific antibodies. Two negative control siRNAs (Control-1, low GC content; Control-2, medium GC content) and two specific siRNAs were used. The asterisk denotes a nonspecific band detected by Protein G-Sepharose beads. Calnexin (CNX) immunoprecipitation served as a loading control. C, effect of SEL1L RNAi on complex formation; the same as B, with the exception that hXTP3-B-long-HA was co-transfected with HRD1-Myc.

FIGURE 10.

Effects of hXTP3-B and SEL1L knock-down on ERAD. A, NHK degradation following XTP3-B siRNA; the same as Fig. 9B, with the exception that NHK was transfected 24 h after RNAi, and cells were pulse-chased 48 h after RNAi, as described in the legend to Fig. 2. One representative result is shown on the left, and quantitative analysis is shown on the right, with error bars indicating the S.E. (n = 3). B, NHK-QQQ degradation following XTP3-B siRNA; the same as in A, with the exception that NHK-QQQ was transfected instead of NHK. C, NHK degradation following SEL1L siRNA. D, NHK-QQQ degradation following SEL1L siRNA.

FIGURE 11.

Involvement of hXTP3-B and EDEM1 in the mammalian ER quality-control pathway. A, effect of co-expression of hXTP3-B and EDEM1 on NHK ERAD. NHK degradation was examined by pulse-chase in cells co-transfected with hXTP3-B-short and EDEM1. Quantitative analysis is shown on the right, with error bars indicating the S.E. (n = 3). B, a model for the mammalian ER quality control scaffold. Only the components analyzed in this study are shown. Both misfolded glycoproteins and nonglycosylated proteins are recruited to the scaffold assembled on the HRD1-SEL1L ubiquitin ligase complex, which also includes hXTP3-B-long, hOS-9, and BiP.