Co-opting the fermentation pathway for tombusvirus replication: Compartmentalization of cellular metabolic pathways for rapid ATP generation
Fig 3
Interaction between the tombusvirus replication proteins and Adh1 fermentation enzyme.
(A) The split ubiquitin-based MYTH assay was used to test binding between the TBSV p33 and the yeast alcohol dehydrogenase Adh1-5p and the Arabidopsis Adh1 proteins in yeast. The bait p33 was co-expressed with the shown prey proteins. The Ssa1p Hsp70 and the empty prey vector (NubG) were used as the positive and the negative controls, respectively. The right panel shows p33: Adh1-5 interactions, the left panel demonstrates that comparable amounts of yeasts were used for these experiments. (B) Co-purification of the yeast His6-Adh1, 2, 3p with the TBSV Flag-p33 and Flag-p92pol replication proteins from subcellular membranes. Top two panels: western blot analysis of the co-purified His6-Adh1-3p with the Flag-affinity purified replication proteins. His6-tagged proteins were detected with an anti-His antibody, while Flag-p33 was detected with an anti-Flag antibody. The negative control was from yeast expressing His6-p33 and His6-p92pol purified using a Flag-affinity column (lane 1). Samples were cross-linked with formaldehyde. Bottom two panels: western blot of the total His6-Adh1-3p and Flag-p33 in the total yeast extracts. (C) Co-purification of the yeast His6-Adh1p with the CIRV Flag-p36 and Flag-p95pol replication proteins from subcellular membranes. See further details in panel B. (D) Co-purification of His6-AtAdh1 with either the TBSV or the CIRV replicase from yeast subcellular membranes. Top two panels: western blot analysis of the co-purified His6-Adh1p (lanes 2–3) with the Flag-affinity purified Flag-p33 or CIRV Flag-p36. His6-Adh1p was detected with an anti-His antibody, while the Flag-p33 or CIRV Flag-p36 replication proteins were detected with an anti-Flag antibody as shown. Bottom two panels: western blot of the total plant protein extracts. (E-F) Pull-down assay including GST-His6-Adh1p or GST-His6-AtAdh1 with the TBSV MBP-p33C or the CIRV MBP-p36C replication proteins and MBP. See further details in panel B. Each experiment was repeated three times.