Figure 4

Purification of the KPAP1 complex. (A) Glycerol gradient fractionation. Mitochondrial extract from T. brucei cells expressing C-terminally TAP-tagged KPAP1 was fractionated on 10–30% glycerol gradients. Fractions were analysed for the presence of RELs (adenylation) and tagged protein (TAP detection). (B) Tandem affinity purification of the KPAP1 complex. The final fraction from the calmodulin column was separated on 8–16% SDS–PAGE and stained with Sypro Ruby. Carryover of TEV protease is indicated by an asterisk. The same fraction was probed along with the mitochondrial extract with antibodies against KPAP1, RET1, and MRP1. Before gel separation, samples were incubated with [-³²P]ATP to detect editing ligases REL1 and REL2. MRP1 has been shown to dimerize even under denaturing conditions (Zikova et al, 2008). (C) PAP and TUTase activities of the purified KPAP1 complex. Left panel: the 89-mer fragment of pre-edited RPS12 mRNA (100 nM) was incubated with KPAP1 complex (10 nM KPAP1, as estimated for the KPAP1-CBP band in Sypro Ruby-stained SDS gel) for 5, 15, and 45 min in the presence of 100 M ATP or UTP, or both. Radiolabelled [-³²P]ATP and [-³²P]UTP were added as indicated. C: RPS12 RNA labelled at the 5' end. Right panel: recombinant KPAP1 (20 nM) and RET1 (20 nM) were incubated with RPS12 RNA under the same conditions. Reaction products were separated on 8% polyacrylamide/urea gel. (D) Synthesis of a short A-tail by the recombinant KPAP1. The 5'-labelled RPS12 mRNA fragment (20 nM) was incubated with 100 M ATP and increasing concentrations of KPAP1 (5, 10, 20, 50, 100, and 200 nM) for 30 min.