Figure 3.

Sir2/4 sub-complex directly interacts with Ubp10 during co-assembly onto chromatin. A, depiction of the domains and protein-binding sites on Ubp10 and Sir4. Ubp10 Cys-371 is the active cysteine involved in deubiquitinase activity. IDR, intrinsically disordered region; USP, ubiquitin-specific protease. The dotted line indicates the broad interaction region on Sir4 that Ubp10 is expected to bind as mapped by previous yeast two-hybrid data (28, 32). B, Western blot analysis of endogenous Ubp10-TAP recruitment to chromatin or Sir2/4-coated chromatin in whole-cell extracts made with sir3Δ. Lane 4, input for both recombinant Sir2/4 and Ubp10-TAP (sir3Δ cells) was combined. C, quantification of B. Signal for Ubp10-TAP is relative to input signal and then normalized to H3 signal. An integration of both a box plot and scatter plot is shown. Error bar indicates at least two independent experiments. D and E, similar experiments performed as in Fig. 2 except recombinant SIR complex is replaced with recombinant Sir2/4 sub-complex. Western blot analysis of recombinant GST-Ubp10 recruitment to pre-bound Sir2/4 chromatin (D) or co-assembly with Sir2/4 onto chromatin (E) is shown. Ubp10 signal in mock reaction quantified as 1 and Ubp10 signal in +SIR complex sample is relative to mock. Mock and relative signals were normalized to H3. Note: image (E) cropped from the same Western blot exposure as depicted in Fig. 2D. Input is as shown in Fig. 2D. F, Western blot analysis of Sir2/4 co-IP of Ubp10. Ubp10 and Sir2/4 were incubated together prior to immunoprecipitation with αCBP-conjugated to protein A beads. G, quantification of F. An integration of both a box plot and scatter plot is shown. Error bar indicates three independent experiments. H, Western blot analysis of GST pulldown of Sir2/4 sub-complex using GST-Ubp10 or GST-Ubp10 Δ109–133 bound to glutathione resin. Sir2/4 was added 1 or 2 times the picomoles of Ubp10 WT or mutant in the reaction.