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.

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.

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.

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.

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.

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 (*).

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 I49 (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).