FIG. 7.

The effect of downregulation and overexpression of WT and mutant Cdc34p on TBSV repRNA accumulation in yeast. (A) Schematic representation of the known domains in Cdc34p. The active-site mutation that abolishes the E2 Ub conjugation activity of Cdc34p is shown with a black box. (B) Northern blotting of repRNA accumulation in yeast with downregulated expression of Cdc34p. The native Cdc34p was expressed from its original location but via the doxycycline (Dox)-regulatable Tet expression promoter. The yeast also expressed a short peptide from pYC2 (as a control), the WT Cdc34p, and the C₉₅S active-site mutant of Cdc34p from low-copy-number plasmid pYC2. Yeast was grown in the absence or presence of 10 μg/ml doxycycline as indicated. Doxycycline was added 6 h prior to launching TBSV repRNA replication. The accumulation level of repRNA was measured 22 h after induction using ImageQuant software. rRNA was used as a loading control (see bottom panel). Western blotting results show the levels of Cdc34p and p33 for the above-described yeast samples. (C) Overexpression of Cdc34p increases repRNA accumulation in yeast. (Top) Northern blotting shows the accumulation of TBSV repRNA in yeast overexpressing of a short peptide from pYC2 or WT Cdc34p in BY4741; (bottom) Western blotting results show the levels of Cdc34p and p33 in the above-described yeast samples. Further details are as described for panel B. (D) Effect of overexpression of WT or mutated His₆-tagged Cdc34p from high-copy-number plasmid pYES on repRNA accumulation in BY4741 yeast. The shown Cdc34p mutants are the E2 site mutant (C₉₅S) and the C-terminally (N170; deletion of aa 171 to 295) and the N-terminally (C125, deletion of aa 1 to 170) truncated versions (Fig. 7A). (Bottom) Western blotting to show the overexpressed His₆-tagged Cdc34p mutants. Further details are as described for panel B.