Figure 5.

S65E is a poor mimetic of pPARKIN. A and B, ST (A) and MT (B) experiments titrating Ub or pUb into a UbFluor reaction with PARKIN^S65E (green triangles) were compared with PARKIN (orange circles) and pPARKIN (blue diamonds) titrations from Fig. 4. Conditions were as described in Fig. 4. Each titration was repeated twice, and all data are shown. Solid lines join the rates determined from one titration experiment, and dashed lines join rates determined from a second titration. C and D, ubiquitination of MiroS by pPARKIN, pPARKIN^C431A, PARKIN, or PARKIN^S65E were performed by the UbMES-based ByS reaction (C) or the native reaction (D). Conditions for these experiments were identical to those used in Fig. 2F. * indicates ubiquitinated MiroS. P indicates PARKIN and M indicates MiroS.