We also FLAG-affinity-purified 66His/FLAG-tagged Ded1p from the membrane-fraction of yeast co-expressing HA-p33 and HA-p92 and found that the purified Ded1p preparation contained HA-tagged p33 ( Figure S2A , lane 1). The affinity-purified Ded1p preparation also showed TBSV replicase activity in vitro on added TBSV template ( Figure S2B , lane 2), suggesting that the membrane-bound Ded1p is associated with the active tombusvirus replicase. Altogether, these data support the model that Ded1p is part of the tombusvirus replicase. Figure 1 . Co-purification of Ded1p with the p33 replication protein from yeast. The FLAG/66His-tagged p33HF or 66His-tagged p33H was purified from yeast extracts using a FLAG-affinity column (lanes 3-4). Top panel: Western blot analysis of Ded1p tagged with 66HA, expressed from the natural promoter in the chromosome, with anti-HA antibody in the purified p33 preparations. Bottom panel: Western blot analysis of HF-tagged p33 or 66His-tagged p33 with anti-His antibody. Note that ''total'' represents the total protein extract from yeast expressing the shown proteins. Each experiment was repeated three times. doi:10.1371/journal.ppat.1002537.g001