Figure 4.

Reconstitution of CCT subunit ubiquitination with purified factors

(A) ³⁵S-methionine-labeled CCT4-3xFLAG was translated in the PURE system and the soluble and monomeric population was isolated (see Figure S5A). This was incubated with E1 and E2 enzymes, His-Ub, ATP, the indicated recombinant HERC2 variant, and ZNRD2, as indicated. The samples were analyzed directly (input) or after denaturing His-Ub pull-down (His-Ub PD).

(B) Each CCT subunit was subjected to in vitro ubiquitination as in (A).

(C) In vitro ubiquitination as in (A), with either His-Ub (WT) or its lysine-free mutant (K0).

See also Figure S5.