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Fig. 2. Mutation in SEM1 causes defects in proteasome function. (A) Genetic interactions between sem1
and proteasome mutations. Synthetic lethality of sem1 with rpn1-821 at 34°C (top) and with pre2-75 at 34°C (bottom) is shown. (B) Suppression of Dsk2-mediated growth arrest by disruption of PRE9. pGAL1-DSK2 was introduced into wild-type, pre9 or congenic sem1 cells. The disruption of PRE9, SEM1 or both genes did not affect the interaction of Dsk2 with the 26S proteasome (data not shown). (C) Accumulation of polyubiquitinated proteins in sem1 mutants. Cell extracts were subjected to immunoblot analysis with anti-polyubiquitin antibody (top) or with anti-Cdc28 antibody (bottom) as a loading control. (D) Defective protein degradation in sem1 cells. The rapidly degraded model substrate Leu-ß-gal and the stable Ala-ß-gal control proteins were expressed as ubiquitin fusions from galactose-induced genes. Transcription was blocked with glucose (0 minute), and proteins were followed for 80 minutes by anti-ß-gal immunoblotting.
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