Figure 5.

Analysis of Mdn1-Dependent Assembly of the Rix-1 Particles in Fission Yeast

(A) Two-hybrid assay for the interaction between S. pombe Mdn1 MIDAS (4301–4717 amino acids [aa]) and S. pombe Rsa4 MIDO domain (1–144 aa, with or without E105D mutation) or S. pombe Ytm1 MIDO domain (1–93 aa).

(B) Wild-type cells, in which wild-type (WT) or mutant (E105D) full-length Rsa4, or vector control (pRep1) were overexpressed from nmt1 promoter, were streaked on plates. Thiamine (Thi) addition represses transcription from nmt1 promoter.

(C–E) Whole-cell extracts (Input) were prepared from wild-type (WT) and mdn1-ts26 (ts) cells (incubated for 6 hr at 36°C) or wild-type cells treated with Rbin-1 (1 μM) for indicated times. Rix1-5FLAG was immunoprecipitated with anti-FLAG antibodies (IP: FLAG). The indicated proteins ([C] GFP-Rsa4, [D] Ytm1-3HA, [E] GFP-Rsa4) and Rix1-5FLAG were analyzed by SDS-PAGE and western blotting. The grouping of images from different parts of the same gel is indicated by dividing lines. The graph in (E) shows relative amount of Rsa4 in IP samples (mean ± range, n = 2).

See also Figure S6.