Fig. 2.

Asr1 associates with the Ubp3/Bre5 deubiquitylase. (A) Asr1 coprecipitates with Ubp3. Extract was prepared from cells expressing HA-tagged Ubp3, either alone (YTM1) or in conjunction with FLAG (FL)-tagged Asr1 (YTM2). Immunoprecipitation (IP) was performed with an anti-FLAG antibody (F-IP), and products were subjected to immunoblotting (IB) with anti-HA and -FLAG antibodies. For Ubp3–HA, 1% of the input (inp) to the IP was also analyzed by IB; for Asr1–FLAG, 7.5% of the input was analyzed. (B) Asr1 coprecipitates with Bre5. Extract was prepared from cells expressing FLAG (FL)-tagged Bre5, either alone (YTM3) or in conjunction with HA-tagged Asr1 (YTM4). IP was performed with an anti-HA antibody (H-IP), and products were subjected to IB with anti-HA and -FLAG antibodies. For Bre5–FL, 1% of the input (inp) to the IP was analyzed by IB; for Asr1–HA, 7.5% of the input was analyzed. (C) The amino terminus of Asr1 is required for association with Ubp3. Extract was prepared from yeast cells expressing galactose-inducible HA-tagged Asr1 proteins, either alone (WT, YTM9; Asr1^RINGm, YTM10; Asr1^PHDm, YTM11; CBD, YTM12) or in the presence of a plasmid expressing MYC-tagged Ubp3 (WT, YTM13; Asr1^RINGm, YTM14; Asr1^PHDm,YTM15; CBD, YTM16). IP was performed with an anti-MYC antibody (MYC-IP), and products were subjected to IB with anti-HA and -MYC antibodies. The * indicates a low molecular weight MYC-reactive species that we assume is a degradation product of Ubp3 that forms during the IP. (D) The amino terminus of Ubp3 mediates interaction with Asr1. Extract was prepared from yeast cells expressing galactose-inducible HA-tagged Asr1 and FLAG-tagged Bre5, either alone (–, YTM9) or in the presence of a plasmid expressing MYC–tagged Ubp3 proteins (WT, YTM13; ∆N180, YTM17; ∆N145, YTM18; ∆N90, YTM19; ∆N45, YTM20). IP was performed with an anti-MYC antibody (MYC-IP), and products were subjected to IB with anti-HA, -FLAG, and -MYC antibodies.