Ime2 promotes mitochondrial detachment through MECA destabilization. pGAL-NDT80 GAL4.ER cells without OsTIR1 (UB17552) and with pCUP1-OsTIR1 (UB17548) as well as pGAL-NDT80 GAL4.ER ime2-as1 cells without OsTIR1 (UB17554) and with pCUP1-OsTIR1 (UB17550) were induced to sporulate. Then, 1 µM β-estradiol and 20 µM 1-NA-PP1 were added to the cultures at 5 h. At 6.5 h, 50 µM CuSO4 and 500 µM 3-indoleacetic acid (auxin) were added. (A) Immunoblot showing Num1-AID-3V5 depletion. Hxk2, loading control. Time indicates hours after addition of auxin and CuSO4 at 6.5 h, a time at which Num1 protein level was already reduced. By band quantification normalized to loading, Num1-AID-3V5 was 2.3× higher in level in ime2-as1 (no OsTIR1) compared with the wild-type control (no OsTIR1). Num1-AID-3V5 runs above the highest (190-kD) ladder band. (B) Maximum-intensity projections of cells fixed 2 h after auxin and CuSO4 addition. Mitochondria, Cit1-GFP; nuclei, Htb1-mCherry. (C) Quantification of the frequency of mitochondrial detachment among cells fixed 2 h after auxin and CuSO4 addition. Analysis was restricted to cells that had entered the meiotic divisions (n = 50 cells per genotype). P = 0.1117, IME2 –OsTIR1 vs. IME2 +OsTIR1, by Fisher’s exact test; P < 0.0001, ime2-as1 −OsTIR1 vs. ime2-as1 +OsTIR1. Scale bar, 2 µm.
