Fig. 4. The TRA-2 MX region is required for the TRA-1–TRA-2 interaction. (A) Amino acid sequence of the TRA-2 MX region. The MX region extends from the cysteine at aa 1392 to the arginine at aa 1413. Amino acids that are identical in C.elegans, C.briggsae and C.remanei are shown in black; amino acids identical in two of these species, including C.elegans, are shown in gray. The amino acids changed in tra-2(mx) mutants are marked by asterisks and include: tra-2(e2021), a C to Y transition at aa 1392; tra-2(e1939), an E to K transition at aa 1393; tra-2(q179) or tra-2(e2019), an R to Q transition at aa 1400; tra-2(e1403), a P to L transition at aa 1411; and tra-2(e1940), an R to Q transition at aa 1413 (Kuwabara et al., 1998). The MX mutants employed in this study are: MXΔ, which removes aa 1392–1412; MX-E1393K, the tra-2(e1939) change; MX-R1400Q, the tra-2(q179) change; and MX-P1411L, the tra-2(e1403) change. Data are presented only for MX-E1393K and MX-R1400Q. (B) MX mutant proteins. Clones encoding GAL4 AD fusion proteins with either wild-type (+) TRA-2ic or one of three mutant TRA-2ic proteins were tested by expression in vitro for generation of protein of the correct size (see Materials and methods). Lanes are as follows: m, molecular weight markers; wt, wild-type AD–TRA-2ic fusion; q179, AD–TRA-2icR1400Q fusion; e1939, AD–TRA-2icE1393K fusion. Molecular weights are indicated on the left. (C) Two-hybrid results with TRA-2ic-MX mutants. Yeast were co-transformed with one plasmid encoding TRA-1A as a GAL4 DNA-binding hybrid and one of three plasmids encoding TRA-2ic or a TRA-2ic-MX mutant as AD hybrids; they were then cultured on SD/–His/–Trp/–Leu with 2.5 mM 3-AT medium at 30°C for 4–5 days. Sector 1, pDB-p53 and pAD-SV40T; sector 2, AD–TRA-2ic; sector 3, AD–TRA-2ic(q179); sector 4, AD–TRA-2ic(e1939). (D) Two-hybrid results with TRA-2icΔ and TRA-2icΔ-MXΔ proteins. Yeast were co-transformed with one plasmid encoding TRA-1A as a GAL4 DNA-binding hybrid and one of four plasmids encoding AD–TRA-2icΔ1 (sector 1), AD–TRA-2icΔ1ΔMX (sector 2), AD–TRA-2icΔ2 (sector 3) or AD–TRA-2icΔ2ΔMX (sector 4); the yeast were then cultured on –His/–Trp/–Leu + 2.5 mM 3-AT medium for 4–5 days. (E) Expression of TRA-2 mutant proteins in yeast. Immunoblot of yeast proteins probed with mouse anti-HA antibody. Lane 1, yeast CG1945; lane 2, yeast CG1945 transformed with pAD-TRA-2icΔ2; lane 3, yeast CG1945 transformed with pAD-TRA-2icΔ2ΔMX. The GAL4AD–TRA-2ic fusion proteins (either wild type or deletion) are indicated by the arrow.