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. 2015 Jan 14;34(4):559–577. doi: 10.15252/embj.201490062

Figure 5.

Figure 5

The TPR domain of Coi12p and Hsp90 are required for the enhancement of scnRNA loading by ATP in cell lysates
  1. Cell lysate from COI12KO cells at 3 hpm was incubated with radiolabeled double-stranded 27-nt RNAs and the different truncated mutants of Coi12p (schematically shown on the top). The Twi1p-containing complex was immunoprecipitated with an anti-Twi1p antibody, co-precipitated RNAs were separated on a denaturing gel, and 27-nt RNAs were detected by autoradiography. The intensities of the bands in the different conditions relative to the wild-type (WT) sample (2nd column) are shown on the top. A representative experiment is shown on the bottom.
  2. Cell lysate from COI12KO cells at 3 hpm was incubated with radiolabeled double-stranded 27-nt RNAs, wild-type (W), or ΔTPR mutant (Δ) of recombinant Coi12p, and with (+) or without (−) ATP and the ATP regeneration system. The Twi1p-containing complex was immunoprecipitated with an anti-Twi1p antibody, and co-precipitated 27-nt RNAs were detected by autoradiography. The intensities of the bands in the different conditions relative to the sample with wild-type Coi12p and without an ATP supply (1st column) are shown on the top. A representative experiment is shown on the bottom.
  3. Cell lysate from COI12KO cells at 3 hpm was incubated with (+) or without (−) hexokinase followed by radiolabeled double-stranded 27-nt RNAs and recombinant wild-type Coi12p. Loaded RNA was analyzed as in (B). The intensities of the bands relative to the sample without hexokinase are shown.
  4. Cell lysate from DCL1KO cells at 3 hpm was incubated with radiolabeled double-stranded 27-nt RNAs and then with (+) or without (−) ATP and the ATP regeneration system, the HSP70 inhibitor PES, and the HSP90 inhibitor 17-AAG. Loaded RNA was analyzed as in (B). The intensities of the bands relative to the sample without ATP and any inhibitors (1st column) are shown on the top.
  5. Cell lysate from COI12KO cells at 3 hpm was incubated with radiolabeled double-stranded 27-nt RNAs, recombinant wild-type Coi12p, with (+) or without (−) ATP, and with (+) or without (−) ATP-γ-S. Loaded RNA was analyzed as in (B). The intensities of the bands relative to the sample with ATP (the 1st lane) are shown on the top.
  6. GST-tagged wild-type Coi12p (GST-Coi12p) or GST-tagged Coi12p lacking the TPR domain (GST-Coi12p-ΔTPR) was incubated with His-tagged wild-type Hsp82p (His-Hsp82p) or His-tagged Hsp82p lacking the C-terminal MEDVD sequence (His-Hsp82p-ΔC) and affinity-purified with glutathione-coupled beads. Proteins before (input) or after (GST pull-down) the purification were analyzed by Western blot using anti-GST (top) and anti-His (bottom) antibodies. The positions of GST-Coi12p, GST-Coi12p-ΔTPR, His-Hsp82p, and His-Coi12p-ΔC are marked with arrowheads.
Data information: In (A–E), the standard deviation (SD) between technical replicates is indicated.