Fig 5.

The extreme C terminus of Rip1 is essential for its function and important for interaction with Mzm1. (A) SDS-PAGE and BN-PAGE (digitonin) immunoblots of isolated mitochondria from WT and rip1Δ cultures expressing YEp RIP1-Myc, vector control (vec), or rip1Δ22 plasmid. Note that on this BN gel the dimeric bc₁ and late core intermediate comigrate, so the band designation includes both III₂ and III₂*, the latter of which denotes the late core intermediate formed during bc₁ complex assembly. The component is clearly the late core intermediate in the vector control rip1Δ cells. (B) Growth assay at 37°C of rip1Δ cultures expressing YCp RIP1, vector control, or the Δ1, Δ4, or Δ6 Rip1 truncation and coexpressing YCp vector control or MZM1-Myc plasmid. (C) SDS-PAGE and BN-PAGE (digitonin) immunoblots of rip1Δ cultures expressing YCp RIP1, Δ1, Δ4, Δ6, or vector control plasmid. (D) Immunoprecipitation of solubilized mitochondria from rip1Δ cultures expressing the plasmids listed in panel C with or without coexpression of YCp MZM1-Myc, using anti-Myc antibody-conjugated agarose beads and showing the total load (L), the final wash (W), and the eluate (E). (E) Detergent-based protein aggregation assay and SDS-PAGE immunoblot of the total load (T), soluble fraction (S), and pellet fraction (P), as described for Fig. 2 except that cultures were grown at 30°C with mitochondria isolated from rip1Δ cultures expressing the same plasmids as in panel C. The exposure of the Rip1Δ6 blot was extended due to limited steady-state levels of this truncation. (F) Assay described in E for a rip1Δ culture expressing the Δ4 truncation, with and without coexpression of YCp MZM1-Myc.