Figure 6.

Ubc5BF62A does not support polyubiquitin chain assembly. Left, autoradiogram of 10-min ¹²⁵I-polyubiquitin conjugation reactions containing 75 nm Uba1, 200 nm Ubc5B or Ubc5BF62A, and 1 nm GST-Nedd4-2 under reducing conditions (lanes 1–5) as described under “Materials and methods.” Right, autoradiogram demonstrating the stoichiometric formation of ¹²⁵I-ubiquitin thioesters in 1-min reactions under non-reducing conditions containing 1 pmol (40 nm) of Uba1 and 1 pmol of Ubc5B or Ubc5BF62A, as described under “Materials and methods.” The positions of mobility standards are shown to the left; the position for free ¹²⁵I-ubiquitin is shown to the right.