α-Actinin interacts with rapsyn. (A) Y190 cells were cotransformed with pGBT10-rapsyn and rapsyn mutants along with pACT2-α-actinin. Transformed yeast cells were seeded in Leu-Trp-His- plates and scored for β-gal activity: (-) no blue after 8 hr, (+) blue after 2 hr. The coiled-coil domain of rapsyn was required for its interaction with α-actinin. (B) Four amino acids at a time were mutated to cysteine starting at the beginning of rapsyn's coiled-coil domain. The mutants were then subcloned into pGBT10 and cotransformed into yeast cells with pACT2-α-actinin. Transformed yeast cells were seeded in Leu-Trp-His- plates and scored as in (A). Amino acids 309–316 and amino acids 321–324 within the coiled-coil domain were necessary for rapsyn's interaction with α-actinin. (C) pGBT10-raspyn was cotransformed with the pACT2-α-actinin constructs into yeast. A linker region composing amino acids 741–757 was required for the interaction with rapsyn. (D) α-Actinin GST-fusion proteins immobilized on glutathione sepharose 4B beads were incubated with lysates from HEK 293 cells transfected with HA-rapsyn. Bead-associated proteins were subjected to SDS-PAGE and immunoblotting (IB) with indicated antibodies. (E) [35S]-labeled rapsyn was generated by in vitro translation in the presence of [35S]-labeled methionine and incubated with bead-immobilized GST-α-actinin. Bound [35S]-labeled proteins were resolved on SDS-PAGE and subjected to autoradiogram. GST α-actinin (653–824) interacted directly with rapsyn.