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. 2009 Jul 1;23(13):1559–1570. doi: 10.1101/gad.524209

Figure 2.

Figure 2.

EAP1, SCP160, and ASC1 are essential components of the SMY2 pathway. (A) Dependency of suppression on EAP1. mps2Δ eap1Δ pRS316-MPS2 cells were transformed with the listed plasmids and tested for growth on SC-Ade and 5-FOA plates. eap1[Y109A], eap1[L114A], and eap1[Y109A,L114A] code for Eap1 proteins with mutations that partially (eap1[Y109A], eap1[L114A]) or totally (eap1[Y109A,L114A]) disrupt binding to eIF4E (Fig. 2D; Ibrahimo et al. 2006). (B) Coimmunoprecipitation of Eap1, Scp160, and Asc1 by Smy2. Extracts from cells expressing SMY2 EAP1-9Myc or SMY2-6HA EAP1-9Myc were immunoprecipitated by anti-HA-coated magnetic beads and analyzed by immunoblotting with the indicated antibodies. (WCE) Whole-cell extract, 10% of the immunoprecipitation input; (IP) immunoprecipitation. (C) Coimmunoprecipitation of Eap1, eIF4E, and eIF4G by Smy2. Extracts from the yeast cells expressing SMY2 EAP1-9Myc or SMY2-6HA EAP1-9Myc were immunoprecipitated by anti-HA-coated magnetic beads and analyzed by immunoblotting with the indicated antibodies. Abbreviations as in B. (D) Coimmunoprecipitation of Smy2 and Eap1 and mutated Eap1 protein that fails to bind to eIF4E. Extracts from yeast cells with the indicated genotypes were immunoprecipitated by anti-HA-coated magnetic beads and analyzed by immunoblotting with anti-HA, anti-Myc, and anti-eIF4E antibodies. Abbreviations are as in B. (E,F) Dependency of suppression on SCP160 and ASC1. mps2Δ scp160Δ pRS316-MPS2 (E) or mps2Δ asc1Δ pRS316-MPS2 cells (F) were transformed with the indicated plasmids and tested for growth on SC-Leu and 5-FOA at 23°C.