FIG. 3.
Analysis of Dia2 mutants. (A) Diagram of Dia2 domains and mutants used in this study. Relevant amino acid residues or changes are indicated. TPR, tetratricopeptide repeats; F, F-box domain; LRR, leucine-rich repeats; NLS, nuclear localization sequence. (B) Expression of Dia2 deletion mutants. Asynchronous strains expressing Dia2Myc (lane 2) or the various mutants (lanes 3 to 6) were grown at 30°C and collected. TCA precipitates were prepared as described in Materials and Methods and immunoblotted with anti-myc or anti-Pgk1 antibodies. Extract from a wild-type strain was included as an untagged control (lane 1). (C) Mutants lacking the F-box do not associate with Skp1. Dia2 mutants were expressed from plasmids using the GAL1,10 promoter and coimmunoprecipitated with anti-Skp1 antibodies. Immunoblots were probed with anti-myc or anti-Skp1 antibodies. The identity of the protein in each band is indicated. Asterisks indicate breakdown products. (D) Dia2 mutants do not rescue dia2Δ phenotypes. Strains expressing integrated Dia2Myc and variants were spotted to YPD plates containing the indicated amounts of hydroxyurea (HU) or MMS in 10-fold dilution series and grown at room temperature.