Fig. 2. An ole1 cDNA or oleic acid suppresses the toxic activity of the WW domain 1 mutant, and the WW domain 1 mutant inhibits ole1 gene expression and Spt23 processing. (A) Cells containing pYes-WW1 were transformed with the ole1 cDNA expression construct (ole1) or an empty vector control (V). Cells were processed as described in the legend of Fig. 1B. For the oleic acid experiment, cells containing pYes-WW1 were plated onto either plain galactose media (-) or galactose media that had been supplemented with 0.5 mM of this unsaturated fatty acid. (B) Cells transformed with pYes (V) or pYes-WW1 (WW1) were picked and processed as described in the Materials and Methods. RNA was isolated from cells, separated on agarose gels containing formaldehyde, transferred to nylon membrane and an ole1 northern blot was performed. Also depicted is a picture of the ethidium-bromide-stained gel prior to transfer showing equivalent loading of RNA. (C) Cells were transformed first with the pESC-FLAG-Spt23 expression construct and then with pYes (v) or pYes-WW1 (WW1). Transformed cells were grown in glucose media overnight. Cells were pelleted and resuspended in galactose media. Cultures were harvested 6 hours later for the preparation of protein extract. Measurement of Nedd4 protein in the extract was performed by a western blot using the anti-Nedd4 polyclonal antibody while the amount of membrane-bound (MB) and processed Spt23 was measured by the anti-FLAG antibody M5. Ponceau S staining of the blot after transfer revealed equivalent loading of total protein.

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