Extended Data Figure 6: The role of the PCI domain and the C-terminal helix in CSN assembly.
a–e, SDS–PAGE analysis of the effects of C-terminal helix and/or PCI domain deletions in CSN subunits: CSN1 (a); CSN2 (b); CSN3 (c); CSN4 (d); and CSN8 (e) on their ability to incorporate into CSN. All complexes contained one Strep(II)-tagged subunit (as indicated) and His6-tags on the other subunits. The cell lysate was divided and subjected to parallel Strep(II)-tag pull-downs using Strep-Tactin beads to probe complex integrity (top) and His6-tag pull-downs using Ni-NTA beads for expression controls (bottom). CSN1 (a) and CSN4 (d) are dependent on the presence of their C-terminal helix (CSN1 isoform 2 residues 466–527; and CSN4 364–406) for integration into CSN. The deletion of C-terminal helix of CSN3 (residues 364–423) impaired its ability to incorporate into the holoenzyme (c). For CSN2 (b) and CSN8 (e), deletion of their C-terminal helices (CSN2 residues 417–443 and CSN8 residues 159–209/166–209) had no effect on complex incorporation and integrity. f, SDS–PAGE analysis showing the inability of C-terminally truncated CSN5 to incorporate into CSN. The co-expressed complexes bore a Strep(II)-tag on CSN6 and His6-tags on the other subunits. g, The CSN (CSN (CSN6ΔMPN)) complex pulled down by the Strep(II)-tagged C-terminal fragment of CSN6 (residues 192–327) was stoichiometric. Arrows indicate truncated subunits. Topological knot between CSN5 and CSN6: h, CSN5 and CSN6 are tightly interlinked. Cartoon representation of the topological knot made by CSN5 (cyan) and CSN6 (orange) immediately before entering the helical bundle. The CSN6 loop (residues 208–214) that crosses CSN5 was disordered in most crystals and is not included in the CSN model.
