Figure 2.

BPA was incorporated at different positions of AHA2 and cross-linked in vivo with UV irradiation. A, whole-cell extract from yeast cells expressing His₆-HA–tagged AHA2 amber mutants and the tRNA synthetase/tRNASup in the absence (−) or presence of 2 mm BPA(+) were analyzed by Western blotting with anti-HA antibody. B, drop test for the growth of yeast strains expressing BPA–AHA2s on plates containing either 2% galactose or 2% glucose + 2 mm BPA at pH ∼6.5. Growth was recorded after 4 days. C, cells expressing BPA-containing AHA2 were kept in the dark (−) or exposed to UV 365 nm (+), and AHA2 was purified from cell extracts using nickel-Sepharose resin, followed by Western blotting with anti-HA antibody. The cross-linked species are observed as higher molecular bands in the upper part of the gel above the monomeric 110-kDa ATPase polypeptide. D, Strep-HA–tagged AHA2s containing BPA was purified with StrepTactin-Sepharose (GE Healthcare) resin and analyzed by NuPAGE Tris acetate gel. On this gel, the cross-link bands are indicated as a dimer and a trimer of AHA2.