Fig. 1. Nedd4 WW domain 1 mutants block the growth of budding yeast. (A) The known functional domains of human Nedd4 and consensus amino acids of the WW domain are depicted. The amino acid underlined is the residue of WW domain 1 that was mutated in the toxic Nedd4 clone as well as the one that was changed to an Ala by site-directed mutagenesis. (B) Cells were transformed with pYes (V), pYes-wt Nedd4 (wt) and the pYes constructs encoding the WW domain 1 point (WW1) or deletion (WW1) mutants. Single colonies were picked from plates and grown overnight to equal densities in glucose media. Cells were diluted to varying degrees and streaked onto glucose and galactose agar plates. Cells were incubated for 3 days. It should be noted that multiple clones from each transformation were analyzed and the results obtained were identical to those presented. (C) Cells transformed with the same constructs presented in Fig. 1B were grown in glucose media overnight. Cells were pelleted and resuspended in glucose (-) or galactose (+) media. Cultures were incubated for 8 hours. Cells were pelleted, protein extract was prepared and the amount of Nedd4 was measured by a western blot. Ponceau S staining of blots after transfer revealed equivalent loading of total protein.