Figure 3: Ubp2 is a degradation substrate of Mdm30.

(a,b) Dextrose and glycerol spot assays at 37 °C (a) or 30 °C (b) of MDM30 (MCY971) and MDM30 ubp2Δ (MCY996) shuffling strains before (WT and ubp2Δ) or after (mdm30Δ and mdm30Δ ubp2Δ) cure of the MDM30 shuffle plasmid. (c–e) MW in kDa are shown on the left of indicated regions of immunoblots. (c) Total protein extracts prepared from ubp2Δ mdm30Δ cells co-expressing Ubp2-HA and WT or f-box mutant Mdm30-Myc, treated (+) or untreated (−) with MG132, were analysed by anti-Myc and anti-Pgk1 immunoblotting (bottom) and quantified relative to WT without MG132 (Top). Error bars represent the s.d. from three independent experiments. (d) Strains from (c) were lysed and lysates were subjected to co-IP with anti-Myc or mock antibody followed by immunoblotting as indicated. Left panels, lysates (10% input of IP); right panels, immunoprecipitates. Red arrow indicates co-IP between Ubp2-HA and the f-box mutant of Mdm30-Myc. Pgk1 was used as a loading and IP control. (e) Total protein extracts from UBP2-6HA (MCY968) and UBP2-6HA mdm30Δ (MCY1031) strains grown for 90 min in YPD (Dextrose) or YPG (Glycerol) in the presence (+) or in the absence (−) of 1 mM CCCP were analysed by anti-HA, anti-Fzo1 and anti-Vph1 immunoblotting. The Mdm30-dependent high MW species of Ubp2-HA are indicated (red arrow). (f) Quantification of CHX chases with UBP2-6HA (MCY968) and UBP2-6HA mdm30Δ (MCY1031) strains analysed by anti-HA and anti-Pgk1 immunoblotting followed by detection with fluorescent secondary antibodies. Error bars represent the s.e.m. from three independent experiments. (g) Dextrose and glycerol spot assays of MDM30 shuffling strains (MCY971) transformed with pRS423-UBP2-6HA (UBP2 o.e.) or an empty vector, before (WT) or after (mdm30Δ) cure of the MDM30 shuffle plasmid.