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. 2022 Sep 6;11:e81755. doi: 10.7554/eLife.81755

Figure 4. H3 binding by Imp5 and sNASP is mutually exclusive and relies on RanGTP for histone transfer.

(A) Imp5-H3 reconstituted complex was purified by size-exclusion chromatography (SEC) and incubated on ice (control) for 3 hr before separating through ultracentrifugation on a 5–40% glycerol gradient. (B) As in (A), reconstituted complex was incubated on ice with equimolar concentration of sNASP (competition assay) for 3 hr before ultracentrifugation. H3 elutes with Imp5, whereas sNASP elutes in its separate fraction. (C) As in (B), but adding Ran in equimolar concentrations to sNASP and Imp-H3. Ran in its purified state is unable to bind to Imp5 and compete with H3. (D) As in (B), but adding RanGTP in equimolar concentrations to sNASP and Imp5-H3 complex. In this instance, RanGTP associates with Imp5 and displaces H3, which co-elutes with its chaperone sNASP. Asterisks indicate an Imp5 degradation product.

Figure 4.

Figure 4—figure supplement 1. Related to Figure 4.

Figure 4—figure supplement 1.

(A) Experimental design for Imp5 purification and complex reconstitution (H3, H4, and H3–H4). SEC280 trace (on Superdex Increase 200), peak volumes (mL), and Coomassie-stained gels. (B) Size-exclusion chromatography (SEC) for sNASP + Imp5 does not resolve these proteins (i.e. elution peaks at 12.6 and 13 mL respectively). (C) 5–40% glycerol gradient ultracentrifugation (240,000 × g, 4°C, 12 hr) sNASP-H3. (D) Imp5 outcompetes pre-formed sNASP-H3 and displaces H3. (equimolar mix).