Fig. 8

Irr1 physically interacts with Imi1. a Interaction of C-terminal part of Irr1 with Imi1 by two-hybrid assay. PJ69-4α strain was co-transformed with plasmids encoding bait and prey proteins as indicated and tested for growth on SD medium lacking histidine in the presence of 10 mM 3-amino-1,2,4-triazole. Plasmids encoding BD-Gal4and AD-Gal4 were used as negative controls [−], whereas a strain bearing BD-Pex1 and AD-Pox5 served as a strong positive control. The interacting fragment corresponds to amino acids from S251 to G344 of Imi1. Interaction with the second identified protein, Mrps5 (fragment comprising amino acids P56-G260), is also shown. b Irr1-GFP and Imi1-RFP co-precipitate. SD-ura-grown cells were subjected to nitrogen starvation for 22 h. Cells were homogenized and proteins were precipitated using magnetic beads coated with anti-GFP or anti-RFP antibodies, as indicated (IP). Immunoprecipitates were separated by SDS-PAGE, transferred to Hybond-C extra membrane and probed with anti-RFP (upper panel) or anti-GFP (lower panel) antibodies. Strains used were: negative controls GFP/Imi1-RFP (lanes 1, 4 and 5) and Irr1-GFP/RFP (lanes 2, 6 and 7), and the study strain Irr1-GFP/Imi1-RFP (lanes 3, 8 and 9)