Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity
Figure 2
(A) UbcH5a (E2) binds to TdT in vitro. His-UbcH5a was incubated with GST-bound (lanes 2 and 4) or GST-TdT-bound (lanes 3 and 5) Glutathione Sepharose 4B in the absence (lanes 2 and 3) or presence (lanes 4 and 5) of His-Ub. Proteins bound to the beads were eluted by boiling with Laemmli buffer. The eluates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-His, anti-TdT, or anti-GST antibody. (B) The pol β-like region in TdT binds to UbcH5a. His-TdT deletion mutants were incubated with GST-bound (lane 2) or GST-UbcH5a-bound (lane 3) Glutathione Sepharose 4B. Proteins bound to the beads were eluted with Laemmli buffer after boiling. The eluates were subjected to SDS-PAGE and analyzed by immunoblotting using an anti-His antibody. (C) E3-independent ubiquitylation of either His-TdT (aa 150–509 or wt) was carried out with UBE1, UbcH5a, and Ub. Ubiquitylated proteins were detected by immunoblotting using an anti-TdT antibody. (D) TdT is poly-ubiquitylated through lysine(s) other than Lys48. TdT was ubiquitylated in a reaction mixture without Ub (lane 1) or containing UbcH5a with His-Ub wt (lane 2), His-Ub K48R (lane 3), Me-Ub (lane 4), or lysine-less (K0) Ub (lane 5). Ubiquitylated His-TdT was detected by immunoblotting using an anti-TdT antibody. (E) In vitro ubiquitylation of TdT mutants. E3-independent ubiquitylation was carried out for wild-type (wt) or point-mutated His-TdTs. TdT was ubiquitylated in the reaction mixture containing UBE1, UbcH5a, and Ub. His-TdT ubiquitylation was detected by immunoblotting using an anti-TdT antibody.