Figure 1. OM14 interacts with NAC.

(a) Scheme of the PCA screen. Yeast strain expressing a bait protein fused to the amino (N)-terminal half of DHFR is mated with a library of yeast strains, each expressing a prey ORF fused to the carboxy (C)-terminal half of DHFR. Mating is done automatically, and the resulting cells are plated in an ordered manner of methotrexate-containing plates. Proximity of the two DHFR halves allows cells’ growth in the presence of methotrexate. Colonies size are determined with Balony program after 3 days of growth and compared with cells that express only the DHFR halves. (b) Example of selected interactions with NAC members, Egd1 and Egd2. Egd2 is known to heterodimerize with Egd1, Tom71 serves as a control for other mitochondria outer membrane proteins and Mck1 is a cytosolic kinase that serves as a standard negative control in such assays. (c,d) Co-immunoprecipitation analysis. Strains expressing either untagged or HA-tagged OM14 were subjected to immunoprecipitation (IP) with anti-HA beads, and samples from different steps of isolation were analysed by western blot with the indicated antibodies. Total indicates sample before mixing with the beads, Flow Thru. (flow through) is a sample from the unbound material, Wash indicates sample from the last wash of beads and IP is the eluted material from the beads. The histogram (d) presents the average results of NAC co-IP efficiency, calculated as the IP/input ratio for NAC divided by HA-OM14 ratio. Data are from at least three independent biological replicates (n), each entailing the entire procedure described above, from cell growth to western analysis. Error bars represent s.e.m. P value=0.028 (independent-samples one-sided t-test). Normal distribution was verified by standard tests (either Shapiro–Wilk or Kolmogrov–Smirnov).