Figure 2. Synergistic functions of Dcn1^P and Hrt1 as Rub1 E3s.

Error bars - +/−1 standard deviation from 3 independent experiments.

A. Phosphorimager data (left) and quantification (right) for time-course of pulse-chase assay monitoring [³²P]-Rub1 transfer from Ubc12 to Cdc53^C+ in the presence or absence of Dcn1^P and the indicated versions of Hrt1 either in complex with Cdc53^C+ or added in trans. To observe comparable levels of Rub1 ligation, a shorter time-course is shown for wild-type Cdc53^C+-Hrt1 in the presence of Dcn1^P.

B. Time course of Ubc12~Rub1 discharge to hydroxylamine as in Fig. 1E, except in the presence or absence of Dcn1^P, for full-length Ubc12 or the isolated core domain (Ubc12core). Raw data – left; quantification – right.

C. Same assay as in (A), but comparing the activity of full-length Ubc12 or the Ubc12 core domain. Raw data – left; quantification – right. Note different times.

D. Same assay as in Fig. 1H, except with earlier time-points and in the presence of Dcn1^P.