FIG. 1.

Interactions between Sup35 and cytoskeletal proteins. (A) Two-hybrid assay. DBD, fusion to the DNA binding domain of Gal4; ACT, fusion to the activation domain of Gal4. “Control” refers to plasmids bearing either Gal4_DBD or Gal4_ACT domains without an insert, respectively. Fusion of the Snf4 protein to the activation domain of Gal4 was used as an additional negative control, showing that Sup35N does not interact with just any randomly chosen yeast protein. Activation of the P_GAL-ADE2 reporter construct resulting from a two-hybrid interaction leads to growth on −Ade medium, shown after 7 days of incubation. In all cases, activation of the P_GAL-HIS3 reporter construct, resulting in growth on −His medium supplemented with 5 mM aminotriazole, was also tested, with the same result (not shown). All Gal4_DBD-Sup35N constructs shown contained Sup35N fused to the N terminus of Gal4_DBD; however, in every case, the same result was also observed with a construct containing Sup35N fused to the C terminus of Gal4_DBD. In all cases where interaction with Sup35N was detected, the same result was observed with complete Sup35. (B) Sla2 protein was immobilized from yeast extracts on resin containing the His-tagged Sup35NM fragment (Sup35NM-His). Yeast extracts were prepared from the [PSI⁺ PIN⁺] yeast strain and run through either Ni²⁺ resin charged with Sup35NM-His protein (after Sup35NM-His) or control resin charged with the protein prepared from the same Escherichia coli strain bearing the control vector instead of the Sup35NM-His overexpressor (control). Eluates were analyzed by SDS-PAGE and Western blotting, followed by reaction to the Sla2 antibody. A similar result was obtained when the yeast strain was transformed with the plasmid coding for HA-tagged Sla2 and reaction to HA antibody was performed (not shown). (C and D) Actin coprecipitates with Sup35-HA (C) but not with Ade2 (D). Proteins were isolated from the [psi⁻ PIN⁺] yeast strain, expressing the HA-tagged Sup35 from the P_CUP1 promoter, and immunoprecipitated with either antibody against HA (recognizing HA-tagged Sup35) (C) or antibody against Ade2 (D). Both total lysates before immunoprecipitation and immunoprecipitates (IP) were run on SDS-PAGE and analyzed by Western blotting, followed by reaction to HA, Ade2, or actin antibody.