Fig. 3.

TRAD1 and HMGB3 are direct CCP1 substrates. A, CCP1 is capable of removing all acidic C-terminal amino acids of TRAD1. The epitope recognized by the PolyE antibody is indicated. B, The C terminus of TRAD1 is processed by active CCP1. Strep/HA- tagged TRAD1 was cotransfected in HEK293F cells either with active CCP1 or inactive (E270Q) CCP1. TRAD1 was enriched from protein extracts using Strep-Tactin columns. Equal amounts of protein eluate were analyzed by Western blot using an anti-Strep antibody. CCP1 is also enriched by means of its C-terminal Strep-tag. The polyE antibody shows the integrity of TRAD1 C terminus when co-expressed with inactive CCP1, but not when co-expressed with the active enzyme. C, In vitro validation of TRAD1 as a direct CCP1 substrate. Purified recombinant TRAD1 was incubated with CCP1 for 2 h at 37 °C, in the presence or absence of the MCP inhibitor o-phenanthroline (o-Phen), and analyzed by Western blot using the polyE antibody. D, HMGB3 is composed of two central DNA binding domains (HMG-boxes) and a long acidic C-terminal tail. CCP1 is able to sequentially release up to 16 acidic amino acids from its C terminus. The size of the HMG boxes and the acidic tail are merely illustrative, and does not represent their real sizes. E, HMGB3 is processed by CCP1. Strep-tagged HMGB3 was cotransfected with active or inactive (E270Q) CCP1. Equal amounts of each cell extract were analyzed by Western blot using a HA-tag antibody, to demonstrate equal expression levels of both CCP1 variants. Western blotting using an anti-Strep antibody shows the appearance of a degraded form of HMGB3 when co-expressed with active CCP1. F, In vitro validation of HMGB3 as a direct CCP1 substrate. Purified recombinant HMGB3 was incubated with CCP1 ON at 37 °C, in the presence or absence of 10 mm o-Phen, and analyzed by SDS-PAGE.