OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD

Abstract

Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant alpha(1)-antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells.

Figure 1. The lectins OS-9 and XTP3-B are ER resident proteins

a. Domain architecture for XTP3-B, OS-9 and Yos9p. Conserved MRH domains (red) and potential N-linked glycosylation sites (Ψ) are indicated. b. Colocalization of endogenous OS-9, XTP3-B and Hrd1 (green) with the ER-resident proteins (anti-KDEL, red) and nuclei (DAPI, blue) by immunofluorescence in HeLa cells. Antibody specificity is presented in Supplemental Information (Fig. S1) c. Separation of OS-9 isoforms by SDS-PAGE and detected by anti-OS-9. 1% Triton X-100 lysates from HEK293 cells untreated (lane 1), + EndoH (lane 2), concanavalin A-Sepharose (ConA) fractions (lanes 3-5) and transiently expressing OS-9.1, 2 & 3 (lanes 6-8). Non-specific background band is indicated by (*). Full scans are presented in Supplemental Information (Fig. S5). d. Transient expression of TCRα-HA in HEK293 cells with endogenous (left) or OS-9 (right) signal sequence. Samples were immunoprecipitated with anti-HA, treated -/+ EndoH, separated by SDS-PAGE and immunoblots probed with anti-HA.

Figure 2. The lumenal domain of SEL1L scaffolds Hrd1, OS-9 and XTP3-B

a. Schematic diagram of full length and truncated SEL1L. Potential N-linked glycosylation sites (Ψ) and Sel-1 domains (triangle) are indicated. b. S-tagged versions of full length (WT), truncated (1-372, 1-737) and KDEL-amended (1-372_KDEL, 1-737_KDEL) SEL1L expressed in HEK293 cells. From normalized protein amounts, coprecipitation of indicated proteins (OS-9, XTP3-B and Hrd1) with representative input controls (20% of starting crude lysate) were assessed by immunoblot with the designated antibodies. c. Coexpression of S-SEL1L_WT with shRNA targeting the indicated OS-9 isoforms and affinity purification as in Fig. 2b. Immunoblots were probed for S-tag and OS-9. shRNA specificity is also presented in Supplemental Information (Fig. S2, S3) d. Pulldowns of SEL1L_WT treated -/+ EndoH and probed for OS-9.

Figure 3. OS-9 and XTP3-B are required for ERAD

a. Coexpression of S-tagged XTP3-B and OS-9.1/2 with NHK-HA. Triton X-100 lysates (LYS, 20%) and anti-HA immunoprecipitates (α-HA) were separated by SDS-PAGE and immunoblots are shown for the S-tag (top), NHK (middle) and tubulin (bottom). Hairline indicates where western blot exposures were joined. Full scans are presented in Supplemental Information (Fig. S5). b. Coexpression of S-tagged XTP3-B with TCRα-HA and RI₃₃₂-HA. Crude lysates (20%) and material coprecipitated by S-protein agarose were probed with anti-HA. Non-specific background band is indicated by (*). c. Representative pulse-chase assay of NHK coexpressed with shRNA (CTRL/GFP, XTP3-B-C, OS-9._1&2, Hrd1-C and SEL1L, left). Bands were quantified by phosphorimager and expressed as the percent of the value at time = 0. Composite data for degradation time courses of NHK coexpressed with shRNA against CTRL (open square), XTP3-B (closed triangle), OS-9.1/2 (open triangle), SEL1L (open diamond) and Hrd1 (closed circle) (right). Data shown represent the mean and S.E.M. from at least 5 individual experiments. The efficacy and specificity of each shRNA are presented in Supplemental Information (Fig. S2, S3) d. Representative pulse-chase assays for the HA-tagged ERAD substrates TCRα and RI₃₃₂ coexpressed with shRNAs targeting XTP3-B and OS- 9.1/2 used in 3c (left). Bar graph (right) represents composite pulse-chase data from at least 3 individual experiments quantified as in 3c. Mean and S.E.M. for 0 and 2 hr. time points are shown.

Figure 4. OS-9/XTP3-B interaction with Hrd1 is mediated through SEL1L

a. Coexpression of NHK-HA, CTRL or SEL1L shRNA and S-tagged XTP3-B, OS-9.1 and OS-9.2 in HEK293 cells. Complexes were affinity purified by S-protein agarose from 1% TritonX-100 lysates. No S-tagged protein was expressed in CTRL lane. Immunoblots are shown for NHK (top), SEL1L (middle) and S-tag (bottom). A lysate input of 20% of the IP is probed for NHK (anti-HA, very bottom). b. Coexpression of S-tagged SEL1L with shRNA (CTRL/GFP, Hrd1-C, XTP3-B-C and OS-9_1&2) in HEK293 cells. Complexes were affinity purified from 1% CHAPS lysates by S-protein agarose. Loading was normalized to approximately equal amounts of S-SEL1L. c. Hrd1-S coexpressed with indicated shRNA and processed as in Fig. 4b. Input lysate representing 20% of the starting material for each affinity purification is shown on left. d. Coexpression of S-tagged XTP3-B and indicated shRNA (described above). In all cases, samples were separated by SDS-PAGE and western blots probed with the indicated antibodies.

Figure 5. XTP3-B and OS-9 interact with ER quality control components

a. S-tagged versions of OS-9.1 & 2, XTP3-B and EDEM1 were expressed in HEK293 cells, lysed in 1% CHAPS and affinity purified by S-protein agarose. Samples were separated by SDS-PAGE and westerns blots performed with antibodies for Hrd1, SEL1L, GRP94 and S-tag to detect coprecipitating proteins. Hairline indicates where western blot exposures were joined. Full scans are presented in Supplemental Information (Fig. S5). b. Immunprecipitations from 1% TritonX-100 detergent lysate of HEK293 cells with anti-GRP94, anti-SEL1L and a control antibody (anti-HA). Western blots were probed with antibodies for OS-9, GRP94 and SEL1L. c. Coexpression of S-tagged OS- 9.1 and OS-9.2 with indicated shRNA (described in Fig. 4b) in HEK293 cells with affinity purification as in Fig. 5a. Western blots were probed with antibodies against S-tag, GRP94, BiP and SEL1L. d. Composite data of pulse-chase assays for NHK coexpressed with a CTRL (open square) or GRP94 (closed triangle) shRNA. Data are presented as in Fig. 3c. Mean and S.E.M. were determined from 4 individual experiments.

Figure 6. Dependence of N-glycan recognition for XTP3-B/OS-9 interaction with ERAD components and substrate

a. Transient expression of S-tagged wild-type, mutant (R207A, R428A, R207A/R428A, N195Q, G379S) and a C-terminal truncation (Δ404-483) of XTP3-B in HEK293 cells, processed as in Fig. 4d and probed for Hrd1, SEL1L and S-tag by western blot. b. Expression of S-tagged wild type and MRH mutant (R188A) of OS-9.1. Immunoblots were performed with antibodies against S-tag, SEL1L and GRP94. c. NHK-HA coexpression with MRH mutants of XTP3-B (R207A/R428A) and OS-9.1 (R188A). Complexes were brought down with anti-HA and immunoblots probed with anti-S-tag (top) and anti-HA (bottom). Hairline indicates where western blot exposures were joined. Full scans are presented in Supplemental Information (Fig. S5). d. HA-tagged transthyretin (TTR-HA) wild-type (WT), D18G and A25T were coexpressed in HEK293 cells with S-tagged XTP3-B and OS-9.1. Complexes were affinity purified by S-protein agarose (top) or anti-HA (bottom) from cells lysed in 1% TritonX-100. Immunoblots were performed with anti-S-tag and anti-HA (TTR). Non-specific background bands are indicated by (*).

Figure 7. Model for coordinating lumenal surveillance with ubiquitination in mammalian ERAD

Nascent glycoproteins interact with the ER-resident chaperone calnexin (CNX) where they are either folded correctly and are released for subsequent trafficking to the Golgi apparatus (1) or in the case of misfolded proteins, re-enter the calnexin cycle in an attempt to facilitate their maturation. If folding remains unproductive, the substrate becomes the target for demannosylation by ER α-mannosidase I to a Man₈GlcNac₂ form (2). The trimmed mannose structure is recognized by EDEM and displaced from the CNX cycle (3). Further demannosylation to a Man₅ or Man₆ form occurs by the actions of either ER resident mannosidases (α-mannosidase I or possibly EDEM3), or in the case of cycling between the ER and the Golgi apparatus, Golgi Mannosidase I (4). By analogy to Yos9p, these lower mannose structures may be part of the signal recognized by XTP3-B and OS-9 (5). OS-9 scaffolds the ER chaperones GRP94 and BiP which may also help to prevent aggregation or facilitate unfolding of the substrate. OS-9/XTP3-B complexes bound to substrate subsequently interact with the lumenal domain of SEL1L (potentially through their MRH domains) which may facilitate the release of substrate in the case of OS-9 (6). XTP3-B forms a stable ternary complex with SEL1L and Hrd1 (7). For OS-9, binding to SEL1L displaces GRP94 and the substrate is transferred to the Hrd1-SEL1L complex (8). The final step is release of OS-9 and dislocation of the substrate by the dislocation apparatus and subsequent ubiquitination in the cytoplasm (9). In an alternative pathway, misfolded, non-glycosylated proteins may also enter this pathway, perhaps through interactions with XTP3-B (10).