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. 2014 Dec 3;89(4):2064–2079. doi: 10.1128/JVI.02719-14

FIG 4.

FIG 4

WW domain of Rsp5p blocks TBSV RNA replication in yeast. (A) Schematic representation of the fusion proteins used for expression in yeast. The WW domain of Rsp5p containing three WW repeats and the p33 sequence were fused as shown. The functional CFP-p33 fusion protein was chosen as control. (B) For the top panel, Northern blot analysis to detect DI-72(+) repRNA accumulation in yeast coexpressing the shown combination of p33 and p92 fusion proteins was performed. The accumulation level of DI-72(+) repRNA was normalized based on 18S rRNA. For the bottom panel, Northern blot analysis of p33 and p92 mRNA levels in yeast was performed. (C) Western blot analysis of total protein extracts with anti-His antibody. (D) For the top panel, Northern blot analysis to detect DI-72(+) repRNA accumulation in yeast coexpressing the shown combination of p33 and p92 fusion proteins was performed. For the middle and bottom panels, Western blot analysis of total protein extracts with anti-His or anti-Flag antibodies was performed. See further details in panels B and C. (E) Membrane association of the various fusion proteins in yeast. Broken yeast cells were fractionated to obtain supernatant (S, soluble fraction) and membrane fraction (P, pellet). Note that the yeast membrane fraction was washed with 1 M NaCl to remove peripheral membrane proteins. Lanes 9 and 10 represent the total, not fractionated, proteins as standards. (F) The WW-domain–p33 fusion protein shows mostly peroxisomal localization in yeast. Confocal laser microscopy images show the subcellular localization of WW-YFP-p33 fusion protein expressed from GAL1 promoter in the BY4741 yeast strain. The peroxisomes were visualized with Pex13p-CFP marker. The merged images show the colocalization of WW-YFP-p33 and Pex13p-CFP marker. Differential interference contrast (DIC) images are shown on the right. Each row represents a separate yeast cell. (G) Peroxisomal localization of YFP-p33 fusion protein. Yeast was grown under similar conditions and images were taken as in panel F. Each experiment was repeated two to three times.