Figure 5.

Growth phenotypes of rsp5-ww domain mutants. (A) Sequence alignment of Rsp5p WW domains. WW1, amino acids 229–266. WW2, amino acids 331–368. WW3, amino acids 387–424. The highly conserved residues of the WW domain are in bold type. The arrow indicates the position of AXXP and FXXA mutations. (B) Expression of HA epitope-tagged ww-AXXP mutant proteins in cells expressing the mutant protein as the sole source of Rsp5p. Cells of the indicated genotypes were grown to early logarithmic phase in SD medium at 24°C. Cells were transferred to YPUAD medium, and aliquots of the cultures were shifted to 30 or 37°C for 15 min. Extracts prepared from these cells were prepared and analyzed by immunoblotting with HA antibodies. The asterisk indicates an unrelated cross-reacting band that serves as a loading control. WT, wild type. (C) ww-AXXP and ww-FXXA mutants are viable. Equal numbers of rsp5Δ cells carrying the indicated RSP5 plasmids were plated onto YPUAD medium (three serial 1:10 dilutions) and grown 3 d at 30°C. (D) Growth of single, double, and triple ww-AXXP mutants. Cells of the indicated genotype were serially diluted as in C and were grown on YPUAD medium at 24°C for 3 d, 30°C for 2 d, or 37°C for 2 d.