RNA–protein interactions promote asymmetric sorting of the ASH1 mRNA ribonucleoprotein complex

She2p mutants N36S and R36K are able to interact with She3p in vitro and in vivo. (A) In vitro GST pull-down assays were performed with 1 μg of (lane 1) GST, (lane 2) GST-She2p-wt, (lane 3) GST-She2p-N36S, or (lane 4) GST-She2p-R63K incubated with a C-terminal domain of She3p prepared by coupled in vitro transcription/translation. (B) She2p mutants N36S and R63K are able to interact with She3p by the two-hybrid assay. Strain PJ69-4a was transformed with the following combinations of plasmids: pGBDU-c1 (vector)/pACT2 (vector), pRL418 (pGBDU-c1-She2p-wt)/pACT2, pGBDU-c1/pEP13 (pACT2-She3p), pRL418/pEP13, pRL419 (pGBDU-c1-She2p-N36S)/pEP13, and pRL431 (pGBDU-c1-She2p-R63K)/pEP13. Transformants were grown in liquid culture and processed for β-galactosidase assays. (C) Characterization of the anti-She2p rabbit antiserum. Extracts were prepared from yeast strains YLM777 transformed with (lane 1) YCplac111 (vector), (lane 2) pRL453 (YCplac111-She2p), and (lane 3) pRL199 (YCplac111-She2p-myc₆). Transformants of YLM777 were grown in synthetic media devoid of leucine. Lysates were prepared and used for Western blotting with anti-She2p rabbit antiserum. In addition, 5 ng of purified (lane 4) GST, (lane 5) GST-She2p-wt, (lane 6) GST-She2p-N36S, and (lane 7) GST-She2p-R63K were analyzed by Western blot with anti-She2p rabbit antiserum. The bands corresponding to She2p, She2-myc₆, and GST-She2p are indicated by the arrows. (D) She2p mutants N36S and R63K associate with full-length endogenous She3p in vivo. Yeast strain YLM1320 was transformed with the following combinations of plasmids: (lane 1) YCplac22 (vector)/pRL453 (YCplac111-She2p-wt), (lane 2) pRL460 (YCplac22-She3p-myc₆)/pRL453, (lane 3) pRL460/YCplac111 (vector), (lane 4) pRL460/pRL462 (YCplac111-She2p-N36S), or (lane 5) pRL460/pRL463 (YCplac111-She2p-R63K). Transformants were grown in synthetic media devoid of leucine as well as tryptophan, and lysates were prepared. An aliquot of the total lysate was analyzed by Western blot for She3p-myc₆ and She2p (total). She3p-myc₆ was immunoprecipitated from the lysate using anti-myc antibody, and the immunoprecipitates were analyzed by Western blot for the presence of She3p-myc₆ and She2p (IP). She2p was detected in the immunoprecipitates using anti-She2 rabbit antiserum. Subsequently, the Western blot was stripped and reprobed with anti-myc to detect She3p-myc₆.