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Fig. 2. Yap8p degradation involves the ubiquitin-proteasome pathway. (A) The erg6
mutant (BY4741 background) was transformed with a plasmid expressing Yap8p-HA. Proteasome activity was reduced by adding MG132 (0.1 mM) to cell cultures and Yap8p-HA levels were monitored as above. Since MG132 was dissolved in DMSO, an equal volume of this solvent was added to the control sample. A long exposure time was needed to visualize Yap8p-HA in the control sample of the erg6 strain. (B) Yap8p levels increase in the pre1-1 pre4-1 mutant that is defective in the 
-type subunits of the catalytic 20S core of the proteasome. The Yap8p-HA plasmid was transformed into wild type (WCG4a background) and the pre1-1 pre4-1 mutant, and Yap8p-HA levels were monitored by western blot analysis. (C) The rate of Yap8p degradation is reduced in cells lacking the ubiquitin-conjugating enzyme Ubc4p. Cells (in BY4741 background) expressing Yap8p-HA were exposed to 0.5 mM As(III) for 1 hour, then washed and placed in growth medium without As(III). Cycloheximide (0.1 mg/ml) was added and Yap8p-HA levels were monitored by western blot analysis. Yap8p protein levels were quantified and normalized to the Hog1p level of each lane.
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