Increased accumulation of TBSV repRNA in ssa1ssa2 mutant yeast after short heat shock treatment. (A) Northern blot analysis of TBSV repRNA accumulation level in ssa1ssa2 mutant yeast cultured continuously at 23°C. TBSV repRNA accumulates to 15% in ssa1ssa2 mutant yeast of that found in wt yeast at the 16 hour time point after induction by galactose. The ribosomal (r)RNA is used as a loading control. (B) After 30 min heat shock treatment of yeast at 42°C, TBSV repRNA replication was launched by expression of p33/p92 and DI-72(+)RNA from the GAL1/GAL10 promoters in ssa1ssa2 mutant yeast by using culture media containing galactose, followed by shaking at 23°C. Northern blot analysis shows that TBSV repRNA accumulated to ~80% in the heat-treated ssa1ssa2 mutant yeast when compared to the wt yeast at the 16 hour time point. (C) Ssa3p can facilitate the in vitro assembly of the TBSV replicase. The purified recombinant FLAG-Ssa3p (lane 2) and FLAG-Ssa1p (lane 1) were added together with purified TBSV p33 (lanes 1-3) and p92^pol (lanes 1-3) to the membrane fraction of a cell-free extract prepared from untransformed yeast, which was programmed via the addition of 1 μg TBSV DI-72 (+)repRNA (lanes 1-3). The newly synthesized repRNA (marked with an arrowhead), representing mostly (+)repRNA due to a full cycle of replication, by the in vitro assembled TBSV replicase is analyzed by denaturing PAGE analysis. The bottom panel shows the coomassie-blue-stained SDS-PAGE gel with the purified recombinant FLAG-Ssa3p and FLAG-Ssa1p.

Cytosolic localization of p33 replication protein in ssa1^ts yeast grown at 36°C. (A) Confocal laser microscopy shows the localization of YFP-p33 in ssa1^ts yeast and SSA1^wt yeast grown at either 23°C or 36°C for 14 hours after induction from GAL1 promoter. The punctate structures show peroxisomal localization, while the “doughnut” -shape structure indicates cytosolic localization of p33 in yeast cells. The punctate structures formed in ssa1^ts yeast grown at 23°C are reminiscent of those found in SSA1^wt yeast (left side of the panel). (B) Cytosolic localization of peroxisome-targeted GFP, termed GFP-SKL, in ssa1^ts yeast grown at 36°C for 14 hours after induction from GAL1 promoter. Note that GFP-SKL carries the peroxisomal targeting sequence “SKL” at the C-terminus.

Ssa1p is required during the in vitro assembly of the TBSV replicase, but not during viral RNA synthesis. (A) A step-wise approach was used to separate the early steps, such as TBSV replicase assembly, from the late steps, which include viral RNA synthesis. The various components used in the procedure are listed. (B) Denaturing PAGE analysis of the radiolabeled in vitro replicase products obtained after the in vitro reconstitution of the TBSV replicase using the conditions shown during step 1 and step 3. (C) Similar analysis of the in vitro TBSV replicase assay with Ssa1p^ts or Ssa1p^wt as in panel B, except using different conditions during step 1 and step 3 as shown. Note that samples in lanes 5-8 contained no Ssa1p^ts or Ssa1p^wt during the replicase assembly.

Time-course experiments to analyze re-distribution of p33 from the cytosol to the peroxisomes after shifting down the temperature from nonpermissive to permissive in ssa1^ts yeast. (A) Confocal laser microscopy shows the localization of YFP-p33 in ssa1^ts yeast grown at 23°C in a media containing glucose (to shut down the production of new p33/p92/repRNA) until the shown time point after switching from 32°C. See the details about yeast culturing in the legend to Fig. 6A. Note that the sizes of the characteristic punctate structures containing YFP-p33 grew over time in ssa1^ts yeast cells. (B) Localization of YFP-p33 in SSA1^wt yeast grown at 23°C in a media containing glucose until the shown time point after switching from 32°C. See further details in panel A. (C) Localization of YFP-p33 in SSA1^wt yeast grown continuously at 23°C in a media containing galactose. (D) Western blot analysis of fractionation experiments shows the increasing extent of re-localization of p33 and p92^pol from the soluble fraction to the membrane fraction in ssa1^ts yeast after shifting down the temperature to 23°C. See the details for yeast culturing in panel A. The fractions were obtained as described in Fig. 6B. (E) Western blot analysis of p33 level in total protein extracts prepared from yeast grown as described in panel A and D.

Lack of TBSV repRNA replication in ssa1^ts yeast at the nonpermissive temperature. (A) Top panels: Northern blot analysis of TBSV repRNA accumulation in ssa1^ts yeast and SSA1^wt yeast grown either at 32°C or 36°C, which inactivates Ssa1p^ts. Bottom panels: Ethidium bromide stained agarose gel shows repRNA and ribosomal rRNA levels in the total RNA samples in ssa1^ts and SSA1^wt yeast strains 16 hours after the induction of TBSV repRNA replication via galactose. Note that YFP-p33 and CFP-p92 each expressed from the GAL1 promoter, whereas DI-72 repRNA was expressed from the GAL10 promoter. (B) Western blot analysis to show the accumulation of YFP-p33 and CFP-p92 in ssa1^ts yeast and SSA1^wt yeast grown at 32°C. The yeast samples were the same as in panel A. (C) An in vitro replicase assay to test TBSV RNA synthesis in ssa1^ts yeast and SSA1^wt yeast grown at 32°C. We obtained the membrane fraction carrying the tombusvirus replicase from ssa1^ts yeast and SSA1^wt yeast grown at 32°C for 16 hours after the addition of galactose to induce TBSV replication. Note that the replicase contains the endogenous repRNA template. (D) Similar in vitro replicase assay was used to test TBSV RNA synthesis in ssa1^ts yeast and SSA1^wt yeast grown at 23°C. We obtained the membrane fraction carrying the tombusvirus replicase from ssa1^ts yeast and SSA1^wt yeast grown at 23°C for 16 hours after the addition of galactose to induce TBSV replication. The bottom panel shows a Western blot for p33 level in each membrane-bound replicase preparations.

The assembled tombusvirus replicase in the membrane fraction of ssa1^ts yeast is not temperature sensitive. Denaturing PAGE analysis shows the radiolabeled repRNAs produced by tombusvirus replicase preparations in vitro obtained from ssa1^ts yeast and SSA1^wt yeast grown at 23°C. The in vitro replication assays were performed at either 25°C or 37°C. The membrane fraction carrying the tombusvirus replicase from ssa1^ts yeast and SSA1^wt yeast grown at 23°C for 16 hours after the addition of galactose to induce TBSV replication was used in the in vitro replicase assay based on the endogenous repRNA template.

Re-distribution of p33 from the cytosol to the peroxisomes after shifting down the temperature from nonpermissive to permissive in ssa1^ts yeast. (A) Confocal laser microscopy shows the localization of YFP-p33 in ssa1^ts yeast grown at 23°C in a media containing glucose (to inhibit the synthesis of new YFP-p33 molecules) for 6 hours after culturing the yeast for 24 hours at 32°C in a media containing galactose. Note that the characteristic punctate structures indicating peroxisomal localization (Jonczyk et al., 2007; McCartney et al., 2005; Panavas et al., 2005a) are formed only in ~50% of ssa1^ts yeast cells. (B) Re-localization of p33 to the membrane fraction in ssa1^ts yeast after shifting down the temperature to 23°C based on fractionation experiments. Disrupted yeast cells were separated to soluble (cytosolic) and membrane fractions via differential centrifugation, followed by Western blotting to detect YFP-p33. Top panel: the samples were taken 6 hours after down-shifting to 23°C (6 hr). Bottom panel: PGK, a soluble yeast protein, and ALP membrane protein were used as internal controls in the Western-blots. See panel A for further details about the growing conditions for yeast samples. (C) Western blot analysis of localization of YFP-p33 in ssa1^ts yeast cells when the samples were taken prior to down-shifting to 23°C (0 hr). Note the partial re-distribution of YFP-p33 from the soluble fraction at the 0 time point (panel C) to the membrane fraction at the 6 hour time point (panel B) in ssa1^ts yeast.

The effect of Ssa1p^ts on the tombusvirus replicase activity. (A) Time-course experiments were performed to analyze the tombusvirus replicase activity in vitro in membrane fractions obtained from ssa1^ts yeast after shifting down the temperature from nonpermissive to permissive. p33/p92^pol replication proteins and DI-72 repRNA were expressed from the GAL1/GAL10 promoters for 16 hours at 32°C, followed by adding cycloheximide (100 μg/ml final concentration, which halted yeast growth) to stop translation and shifting the temperature to 23°C to activate Ssa1p^ts. Top panel: Denaturing PAGE analysis of in vitro replicase products obtained using the membrane-enriched fraction carrying the co-purified repRNA. The activity of the tombusvirus replicase (lanes 15-16) obtained from ssa1^ts yeast grown continuously at 23°C in the presence of cycloheximide was taken as 100%. Middle panel: Western blotting of YFP-p33 present in the membrane fractions used for the in vitro replicase assay in the top panel. Bottom panel: Western blotting of YFP-p33 present in the total protein extract that was used to adjust the amount of replication proteins in each sample. (B) Denaturing PAGE analysis of in vitro replicase products obtained using the membrane-enriched fraction carrying the co-purified repRNA. The replicase preparations were obtained from ssa1^ts yeast grown for 16 hours at 32°C in a media containing galactose, followed by changing to a media containing glucose to stop translation of p33/p92^pol and shifting the temperature to 23°C to activate Ssa1p^ts. See further details in panel A. (C) In vitro replicase assay with tombusvirus replicase preparations obtained from ssa1^ts yeast grown for 16 hours at 32°C in a media containing galactose, followed by shifting the temperature to 23°C to activate Ssa1p^ts. Note that p33/p92^pol are produced continously under this condition. The image shows the denaturing PAGE analysis of in vitro replicase products obtained using the membrane-enriched fraction carrying the co-purified repRNA. See further details in panel A. The level of RNA synthesis by the replicase preparation obtained from yeast 30 min after the down-shift is taken as 100%.