Fig. 2
From: HTLV-1 Tax plugs and freezes UPF1 helicase leading to nonsense-mediated mRNA decay inhibition
HTLV-1 Tax interacts directly with UPF1-HD and inhibits its ATPase activity. a Schematic diagram showing the UPF1 and Tax domains and sites of mutant derivatives used for this study. Structural domains are represented by rectangles and the protein truncations used by black lines. b Pulldown experiment using GST-Tax (lanes 1–6) or GST-His tag (lanes 7–12) as bait. After incubation, protein mixtures before (input 20% of total) or after precipitation (precipitate) were separated on a 10% SDS-PAGE gel and visualised by coomassie staining. c A SDS-PAGE gel illustrating the co-purification of human UPF1-HD-His and HTLV-1 GST-Tax proteins by two sequential affinity purifications (Nickel and Glutathione columns). Samples from the Escherichia coli lysate (lane 1: L), flow through (lanes 2 and 4: FTHIS and FTGST) and eluate (lanes 3 and 5: EHIS and EGST) fractions were loaded on the gel. The purity of the fractions was evaluated by coomassie staining (upper panel). The proteins were visualised by western blot analysis using anti-His and anti-Tax antibodies targeting UPF1 HD-His and GST-Tax, respectively (lower panels). d Graph showing the percentage of [α-32P]ATP hydrolysed as a function of time by UPF1-HD (continuous line, diamond), UPF1-HD/Tax CACA (continuous line, square), UPF1-HD/Tax (dotted line, circle) and Tax CACA (dashed lane, cross) under conditions of steady-state ATPase turnover. Aliquots from the reaction mixture were quenched 0, 1, 5, 10 and 15 min before TLC chromatography (see Methods). The values are the mean of at least three biological replicates, and the error bars correspond to the SD. e UPF1-HD, UPF1-HD/Tax CACA complex and BSA proteins were spotted onto a nitrocellulose membrane and exposed to [α-32P]ATP (top panel). The levels of the proteins used were also analysed by slot-blot on a different membrane. The membrane was incubated with anti-His and anti-Tax antibodies (middle and lower). Nonsignificant lanes were removed as indicated by the vertical white line. Uncropped scans related to Fig. 2 are available in Supplementary Fig. 6
