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. 2016 Feb 10;12(2):e1005440. doi: 10.1371/journal.ppat.1005440

Fig 13. Cof1p and Act1p mutations affect the subversion of MCS forming OSBP-like Osh and VAP proteins for tombusvirus replication in yeast.

Fig 13

(A) Enhanced co-purification of yeast Osh6p ORP with the p33 replication protein from act1-121 ts yeast in comparison with WT yeast. The FLAG-tagged p33 and FLAG-p92 were co-purified from solubilized membranous fraction of yeast extracts using a FLAG-affinity column. Yeast expressing the His6-tagged p33 was used as a control. Top panel: Western blot analysis of the co-purified 6xHis-tagged Osh6p with anti-His antibody in the affinity-purified preparations. Middle panel: Western-blot analysis of the same samples as in the top panel, but using anti-FLAG antibody. Bottom panels: Western blot analysis of 6xHis-Osh6p with anti-His antibody in the total protein extract from yeast expressing the shown proteins. (B) Enhanced co-purification of yeast Osh6p ORP with the p33 replication protein from cof1-8 ts yeast in comparison with WT yeast at the permissive and semi-permissive temperatures. See further details in panel A. (C) Enhanced co-purification of the ER-localized Vap27-1 with the p33 replication protein from act1-121 ts yeast in comparison with WT yeast at the semi-permissive temperature. The FLAG-affinity purification was done as in panel A. Top panel: Western blot analysis of the co-purified 6xHis-Vap27-1 with anti-His antibody in the affinity-purified preparations. Middle panel: Western-blot analysis of the same samples as in the top panel, but using anti-FLAG antibody. Bottom panels: Western blot analysis of 6xHis-Vap27-1 with anti-His antibody in the total protein extract from yeast expressing the shown proteins. CB: Coomassie-stained SDS-PAGE of total protein extract. Each experiment was repeated two times. (D) Enhanced co-purification of the ER-localized Vap27-1 with the p33 replication protein from cof1-8 ts yeast in comparison with WT yeast at the permissive and semi-permissive temperatures. See further details in panel A. (E) Dependence of TBSV repRNA replication on Scs2p VAP protein in yeast. The double-mutant yeast carrying act1-121 ts mutation and missing SCS2 shows reduced replication of TBSV repRNA based on Northern blot analysis. See further details in Fig 1.