Fig. 4: Binding of VHH12G2 to Ube2G2 does not significantly affect its ability to form a Ub thioester, but reduces its ubiquitination activity in the presence of E3s.
A Schematic representation of the ubiquitination cascade. The E1 enzyme activates ubiquitin (Ub) in the presence of ATP and transfers it to the active site cysteine (-SH) of an E2 enzyme (Ub loading). Subsequently, an E3 enzyme with a RING/U-box domain facilitates the transfer of Ub directly from the E2 to the substrates. B Thioester Ub loading assays of Ube2G2 in the presence and absence of VHH12G2, analyzed by SDS-PAGE under non-reducing (left, -DTT) and reducing (right, +DTT) conditions. Reaction mixtures contained human E1 (Uba1), human Ube2G2, human Ub, and VHH12G2 added at a 3-fold molar excess over Ube2G2. Samples were collected at the indicated time points. Thioester Ub complexes detected under non-reducing conditions are labeled as Ube2G2Ub1/2/X based on the number of Ubs attached. SDS-PAGE under reducing conditions destroys any Ub thioesters and shows Lys-linked Ube2G2-Ub adducts. C–E Immunoblots of ubiquitination assays performed with the indicated E3 domains: (C) RING domain of human HRD1 (GST-HRD1), (D) RING domain of human TRC8 (GST-TRC8), and (E) full-length human CHIP (U-box type E3). Immunoblots with anti-Ubiquitin (top), anti-HA (middle, to detect HA tagged VHH), and anti-Ube2G2 (bottom) are shown for (C–E), respectively. Identifiable ubiquitinated species are indicated next to the blots where possible. Ub/Ub2 denotes unanchored ubiquitin.
