Supplementary Figure 1: The carboxyterminus integrates Der1 into the HRD-ligase.
From: Der1 promotes movement of misfolded proteins through the endoplasmic reticulum membrane
a, Sequence alignment of yeast Der1 with homologues in other organisms, generated by ClustalW and Jalview. Derlin-1 from Homo sapiens (hs, UniProtKB accession number Q9BUN8), Caenorhabditis elegans (ce, Q93561), Derlin-2 from Homo sapiens (hs, Q9GZP9), Caenorhabditis elegans(ce, Q21997), Derlin-3 from Homo sapiens (hs, Q96Q80), Mus Musculus (mm, Q9D8K3), Der1 from Saccharomyces cerevisiae (sc, P38307). The position of the Der1 transmembrane segments as predicted by hydrophobicity calculations and biochemical analysis by Hitt et al.16 is given by green bars. Of note, in an alternative model for Derlin-1 topology proposed by Greenblatt et al. 32, the position of transmembrane segments one and two is almost identical. Black diamonds label amino acids in Der1, which were subjected to site-directed mutagenesis (Supplementary Table 1). b, Cycloheximide decay assay to monitor the degradation of Der1 in strains of the indicated genotypes. c, As in b but Δder1 cells were transformed with low-copy number plasmids encoding mutants of Der1. d, Cycloheximide decay decay assay to determine turnover of Der1-Myc and Der1 in Δusa1 cells. The integral ER-membrane protein Sec61 served as loading control. e, Plasmid-encoded HA-tagged Der1 was expressed with endogenous Der1 in cells containing or lacking Usa1. Membranes of the total extract were solubilised with Digitonin and Der1-HA was precipitated with anti-HA antibodies followed by SDS-PAGE and immunoblotting.
