FIG. 5.

Comparison of the effects of RSP5 expression in wt (R1158) and RSP5/THC (TET-RSP5) yeast on the replicase activity and p33 ubiquitination. (A) In vitro replicase assay based on the copurified replicase and the endogenous repRNA in membrane-enriched fractions prepared from R1158 (wt) and RSP5/THC yeast grown with or without doxycycline. Top panel: the denaturing PAGE assay shows the levels of repRNA synthesis by the replicase in vitro, quantified as mean percentages and standard deviations by setting the activity in wt yeast without doxycycline as 100%. Note that comparable amounts of membrane-bound replicase (based on the level of p33) were used in this assay, as shown in the bottom panel. The level of six-His-tagged p33 in the replicase preparations was detected by Western blot analysis. The panel shows experiments performed in duplicate. (B) Ubiquitination of the six-His- and FLAG-tagged p33 protein was tested via Western blotting using anti-FLAG antibody (top panel) and anti-c-myc antibody (bottom panel). Note that monoubiquitination causes an ∼8-kDa shift in p33 protein migration. The membrane-bound p33 was purified via FLAG affinity chromatography after solubilization of yeast coexpressing c-myc-tagged ubiquitin from a plasmid. Ub, ubiquitin; +, present; −, absent.