Fig 10. Recruitment of the cellular Osh/ORP proteins is facilitated by Fis1 into tombusvirus replication compartment.
(A) Co-purification of the yeast His6-Osh6p with TBSV Flag-p33 and Flag-p92pol replication proteins from subcellular membranes of yeast over-expressing Fis1p. Top two panels: western blot analysis of co-purified His6-Osh6p detected with anti-His antibody, while Flag-p33 was detected with anti-Flag antibody. The negative control was from yeast expressing His6-p33 purified in a Flag-affinity column (lane 1). Samples were cross-linked with formaldehyde. Bottom two panels: western blot of total His6-Osh6p and Flag-p33 in the total yeast extracts. (B) Co-purification of the yeast His6-Osh6p with TBSV Flag-p33 and Flag-p92pol replication proteins from subcellular membranes of WT or fis1Δ yeasts. See further details in panel A. (C) Interactions between cYFP-AtFis1A/B and nYFP-AtORP3A proteins within the TBSV p33-BFP-decorated tombusvirus replication compartment were detected by BiFC. Expression of the above proteins from 35S promoter was done after co-agroinfiltration into N. benthamiana leaves infected with TBSV. Scale bars represent 10 μm. Each experiment was repeated. (D) Decreased level of re-localization of ergosterols to internal punctate structures in fis1Δ yeast replicating TBSV (third panel from left) in comparison with WT yeast (bottom panel) or fis1Δ yeast expressing Fis1p from a plasmid (right panel). Fluorescent microscopic images of yeast cells stained with the filipin dye. Note that filipin stains ergosterols present mostly at the plasma membrane in virus-free cells, as shown. (E-F) Confocal laser microscopy images show the lack of co-localization of TBSV GFP-tagged p33 replication protein with the RFP-tagged Stt4p PI4K kinase protein in fis1Δ and their co-localization in WT yeast cells, respectively. DIC and merged images are shown on the right. Note that yeasts co-expressed p92 and the (+)repRNA to support TBSV replication. Scale bars represent 5 μm. Each experiment was repeated.