Supplementary Figure 4: Der1 is in close proximity to dislocated CPY^*.

a, Photoreactive probes were placed at the indicated positions in Der1-Myc. The constructs were expressed in Δder1 Ubc7C/S CPY^*-HA cells either containing or lacking Usa1 and exposed to UV light. The samples were then lysed and subjected to immunoprecipitation as described in Fig. 3. b, Der1-HA expressed from high-copy plasmid pMM079 was transformed into Δder1 Ubc7C/S cells either containing or lacking Usa1. Der1-Myc labelled with pBpa at position G38 (derived from pMM075) was co-expressed where indicated. Cells were lysed in Digitonin containing buffer and Der1-Myc was immunoprecipitated with anti-Myc antibodies. Interacting Der1-HA was detected by immunoblotting. c, Δder1 Ubc7C/S cells were transformed with a low-copy plasmid encoding Der1-HA. Microsomes of these cells were solubilised with NP40 and Der1-HA was precipitated with anti-HA antibodies. The catalytically inactive Ubc7 mutant (Ubc7C/S) was used to adjust the substrate levels in the individual strains. d, Der1-Myc constructs with photoreactive probes placed at positions which reveal prominent crosslinks with Hrd1 were expressed either in Δder1CPY^*-HA cells (wt) or in Δder1CPY^*-HA Ubc7C/S cells. The photocrosslinking was performed as described. e, As in d but the Der1-Myc constructs contained photoreactive probes at positions, which formed crosslinks with Usa1. f, Determination of the unfolded protein response (UPR) in strains used for the crosslinking experiments by β-galactosidase activity assay. The indicated yeast strains were transformed with the pUPRE-lacZ plasmid and the activity of β-galactosidase was measured as described (see Methods). Where indicated cells were treated with 4 mM Dithiotriol (DTT) for 1 hour before β-galactosidase measurement to fully induce the UPR. Error bars and mean values of three independent experiments are shown. g, Der1-Myc variants labelled at the indicated positions were expressed in Δpep4 Ubc7C/S cells containing plasmid-encoded PrA*-HA. The crosslinking experiment was performed as in a.