Supplementary Figure 3: The pBpa-labelled Der1 variants are properly integrated into the HRD-ligase.

a, Der1-Myc labelled at positions in the first (W19) and second (S70) transmembrane domain as well as in the first luminal loop (G38, Y42, L46, K50) (derived from pMM075) were expressed in Δder1 Ubc7 C/S CPY^*-HA cells to investigate crosslinking to Yos9. b, Efficiency of the CPY^*-HA crosslinking to different positions in Der1-Myc. Photoreactive probes were introduced at the indicated positions of Der1-Myc (derived from pMM075) and the crosslinking experiment was performed as described (see Methods). Crosslinked CPY^*-HA and precipitated pBpa-labelled Der1-Myc were detected by fluorescently labelled secondary antibodies using the Odyssey near-infrared scanner (Li-Cor) and quantified by Odyssey Imaging System Version 3.0. The amount of the CPY^*-HA crosslinking at position G38 was set to 100%. The efficiency of the CPY^*-HA crosslinking at other positions was calculated in relation to position G38 and normalised by the corresponding precipitated pBpa-labelled Der1-Myc variant. The asterisk denotes a cross-reactivity of the anti-Myc antibody. c, Pulse chase assay to analyse the activity of pBpa-modified Der1 variants in the degradation of CPY^*. The selected Der1 constructs (derived from pMM063) form prominent crosslinks with different components of the HRD ligase as well as CPY^*-HA and were expressed on high-copy plasmids in Δder1 cells. As a control unlabelled Der1 was expressed on a low-copy (wt) and high-copy plasmid (Der1 OE), respectively.d, Δder1 Ubc7C/S CPY^*-HA cells expressing either various pBpa-labelled Der1-Myc constructs or unlabelled Der1-Myc were lysed in Digitonin containing buffer and subjected to immunoprecipitation with anti-Myc antibodies. Interaction partners of Der1-Myc were analysed by SDS-PAGE and immunoblotting. The asterisk denotes a cross-reactivity of the anti-Hrd1 antibody in the total lysate.