Domain structures of some ERp57 substrates. The domain structures (A–D) common to distinct substrates of ERp57 are illustrated. Proteins listed are substrates of ERp57 that contain domains characterised by little secondary structure and several disulphide bonds, where the number in brackets indicates the number of times the domain occurs in that protein. In the sequences shown, C denotes a cysteine residue, X is any other amino acid and lines represent intradomain disulphide bonds.

ERp57 forms mixed disulphides with Ero1Lα in vivo. Untransfected HT1080 cells (lane1) and stable cell lines expressing ERp57-V5 (lane 2), ERp57-V5 Cys2 (lane 3), ERp57-V5 Cys7 (lane 4), ERp57 Cys2,7 (lane 5) or ERp57-V5 Cys1,2,6,7 (containing no active site cysteines; lane 6) were analysed for mixed disulphides between ERp57 and Ero1Lα. Cells were lysed in the presence of NEM to preserve disulphide status and V5-ERp57 was immunoisolated with an anti-V5 antibody. The resulting samples were separated under reducing (A) and non-reducing conditions (B, C). Proteins were transferred to a nitrocellulose membrane and probed with an anti-Ero1Lα antibody (A, B) or stained with Ponceau S (C).

Oxidative folding of clusterin can occur in the absence of an interaction with calnexin or calreticulin. (A, B) Clusterin was translated in vitro in the presence of reticulocyte lysate, radiolabelled amino acids, SP-MF cells and in the absence (lanes 1–4) or presence of 1 mM castanospermine (lanes 5–8). Translation proceeded for 5 min before initiation was prevented by the addition of ATCA. Following incubation for times as specified, disulphide exchange was prevented by the addition of 25 mM NEM. Proteins were separated by SDS–PAGE under reducing (A) and non-reducing conditions (B) and visualised by exposure to film. G, glycosylated clusterin; C, cytosolic clusterin; ^* indicates potential mixed disulphides. (C) Clusterin was translated in HT1080 human fibroblast cells expressing ERp57-V5 in the absence or presence of 1 mM castanospermine, as in panels A and B. Following treatment with NEM, samples were analysed directly (lanes 1 and 6) or ERp57 was immunoisolated with an anti-V5 antibody (lanes 2–5, 7–10). Samples were separated by SDS–PAGE under non-reducing conditions. MD, mixed disulphides. (D) Clusterin was translated for 60 min in HT1080 human fibroblast cells expressing ERp57-V5 in the absence or presence of 1 mM castanospermine, as in panels A and B. Following treatment with NEM, samples were analysed directly (lanes 1 and 3) or ERp57 was immunoisolated with an anti-V5 antibody (lanes 2 and 4). Samples were separated by SDS–PAGE under reducing conditions.

Clusterin requires ERp57 for efficient oxidative folding. (A, B) Clusterin was translated in vitro in the presence of reticulocyte lysate and radiolabelled amino acids and in the absence (lane 1) or presence of SP-MF cells (lanes 2–5). Translation was stopped after 1 h by the addition of 25 mM NEM. Following lysis, proteins were treated with Endo H to remove glycans (lane 3). Samples were subjected to digestion with proteinase K before (lane 4) or after solubilising with Triton X-100 (lane 5). Samples were separated by SDS–PAGE under reducing (A) or non-reducing conditions (B) before being exposed to film. (C, D) Clusterin was translated as in panels A and B in the presence of either wt SP-MEF cells (lanes 1, 3, 5 and 7) or ERp57^−/− SP-MF cells (lanes 2, 4, 6 and 8). Translation was allowed to continue for 5 min before the addition of ATCA to prevent initiation. Reactions were allowed to proceed for the indicated times and were terminated by adding NEM and placing on ice. Cells were isolated and samples separated under reducing (C) or non-reducing conditions (D). ^* indicates potential mixed disulphides. (E) Clusterin was translated for 16 min in the presence of ERp57^−/− SP-MF cells. Cells were isolated and samples separated under non-reducing (lane 1) or reducing (lane 2) conditions.

ERp57 is essential for the efficient oxidative folding of β1 integrin. (A, B) β1 integrin was translated in the presence of reticulocyte lysate, radiolabelled amino acids and either wt SP-MF cells (lanes 1, 3, 5 and 7) or ERp57^−/− SP-MF cells (lanes 2, 4, 6 and 8). Translation proceeded for 5 min before initiation was prevented by the addition of ATCA. Following incubation for times as specified, disulphide exchange was stopped by the addition of 25 mM NEM. Proteins were separated by SDS–PAGE under reducing (A) and non-reducing conditions (B) and visualised by exposure to film. (C, D) β1 integrin was translated as in panels A and B in the presence of SP-MF cells in the absence (lanes 1, 3, 5 and 7) or presence of 1 mM castanospermine (lanes 2, 4, 6 and 8). Samples were separated under reducing (C) or non-reducing conditions (D). (E) Either intact HT1080 cells were radiolabelled for 30 min and chased with cold media for 30 min to allow correct folding and disulphide bond formation (lane 1) or translation of β1 integrin was carried out as in panels A and B for 120 min in the presence of either wt MF (lane 2) or ERp57^−/− (lane 3) SP-cells. Cells were lysed in the presence of NEM. β1 integrin was immunoisolated from intact cells with an anti-β1 integrin antibody (8E3) and separated by SDS–PAGE under non-reducing conditions. Radiolabelled proteins were visualised by autoradiograph.

ERp57-V5 Cys2,7 forms mixed disulphides when expressed in HT1080 cells. (A) HT1080 human fibroblast cells stably transfected with ERp57-V5 were fixed with methanol. Cells were labelled with a rabbit anti-PDI antibody that was detected with an Alexa Fluor 594 anti-rabbit secondary antibody, a mouse anti-V5 antibody that was detected with an Alexa Fluor 448 secondary antibody and 1,4-diazobicyclo(2,2,2)octane (DAPI). (B) Untransfected HT1080 human fibroblasts (lanes 1 and 4), those expressing ERp57-V5 (lanes 2 and 5) or ERp57-V5 where the active sites had been mutated to CXXA (ERp57-V5 Cys2,7; lanes 3 and 6) were treated with 25 mM NEM and lysed. Clarified lysates were separated under non-reducing (lanes 1–3) and reducing conditions (lanes 4–6). Proteins were transferred to a nitrocellulose membrane and probed with an anti-V5 antibody. (C) HT1080 cells expressing ERp57-V5 Cys2,7 were treated with NEM and lysed. Clarified lysates were immunoisolated with an anti-V5 antibody conjugated to agarose beads. Proteins were eluted by boiling in SDS and separated under non-reducing conditions. Gel lanes were excised and reduced with 50 mM DTT and separated in a second dimension. Proteins were visualised by silver staining. Proteins migrating faster in the second dimension than the first dimension were excised from the gel and identified by mass spectrometry. (D) HT1080 cells expressing ERp57-V5 Cys2,7 were untreated (lanes 1 and 3) or treated with 1 mM castanospermine (CST) (lane 2). Cells were radiolabelled for 30 min and chased with cold media for 30 min, where castanospermine was present throughout in treated samples. Samples were then left untreated (lanes 1 and 2) or treated with DTT (15 mM) for 5 min (lane 3). Cells were lysed in the presence of NEM. ERp57 and mixed disulphides were immunoisolated with an anti-V5 antibody conjugated to agarose beads and separated by SDS–PAGE under reducing conditions. Radiolabelled proteins were visualised by autoradiograph.