Fig 2.

Rip1 and Mzm1 form a complex of low molecular mass, whose formation is dependent upon tyrosine-11 of Mzm1. (A) BN-PAGE analysis of mitochondria isolated from Δbcs1 cells expressing both YCp RIP1 and YCp Sod2-MZM1-Myc, followed by immunoblot (anti-Rip1). (B and C) Subsequent analysis of the complex isolated through BN-PAGE by SDS-PAGE in a second dimension, followed by immunoblot (B, anti-Rip1; C, anti-Myc). The approximate positions of molecular masses are indicated, and a line is drawn as an aid to the eye indicating the Rip1-Mzm1 complex. (D and E) Immunoprecipitation of solubilized bcs1Δ rip1Δ mitochondria with anti-Myc antibody-conjugated agarose beads, showing the load (L), the final wash (W), and the eluate (E). Mitochondria were isolated from cultures expressing YCp RIP1 and either YCp Sod2-MZM1-Myc (D) or YCp Sod2-MZM1-Myc with a mutation resulting in conversion of tyrosine-11 to alanine (E).