Complementation of erh1Δ mutation and cross-species experiments.
(A) Serial dilutions of cells were spotted on EMM2+ADE alone or EMM2+ADE supplemented with 2 M sorbitol, 10 mM hydroxyurea or 0.005% SDS agar plates and incubated for 5 days. Strains used: auxotrophic (ade− leu−) erh1+ FY7269 transformed with pREP1 (ZBM1023), FY7269 erh1Δ derivative ZBM1020 transformed with pREP1 (ZBM1024), ZBM1020 transformed with pREP1/SpErh1p (ZBM1025), ZBM1020 transformed pREP1/SjErh1p (ZBM1026) and ZBM1020 transformed with pREP1/HsERH (ZBM1027). (B) Intracellular localization of SpErh1p in human HeLa cells by confocal microscopy. Upper series of images, cells transfected with plasmid coding for EGFP-tagged human ERH (pEGFP-N1/ER) alone or cotransfected with plasmid coding for mCherry-tagged human Ciz1 (pmCherry-N1/Ciz1); lower series of images, cells transfected with plasmid coding for EGFP-tagged SpErh1p (pEGFP-N1/SpErh1p) alone or cotransfected with pmCherry-N1/Ciz1. Direct fluorescence of EGFP or mCherry was observed in live cells. (C) Yeast two-hybrid analysis with SpErh1p used as bait. The host S. cerevisiae L40 cells coexpressing human ERH and Ciz1, human ERH and PDIP46/SKAR (both pairs as positive controls), SpErh1p and Ciz1, and SpErh1p and PDIP46/SKAR were lysed and the activity of the lacZ reporter gene (conversion of X-gal to a blue precipitate represented here as a strong gray color of lysed cells) was determined.