(A) Viability loss of Smc3p interface mutant strains. Haploid SMC3-AID strain alone (VG3651-3D) or containing either wild-type (WT; BRY474), smc3-I1026R (VG3905-7A) or smc3-L1029R (BRY492) were grown and plated as described in Figure 2. (B) Viability loss of Mcd1p interface mutant strains. Haploid MCD1-AID strain alone (VG3902-3A) or containing, either wild-type (WT; VG3914-2C), mcd1-L75K (VG3916-5B) or mcd1-L89K (VG3918-9D) were grown and plated as described in Figure 2. (C–D) Haploids in A and B were synchronously arrested in mid-M phase as described in Figure 3. The number of GFP spots was scored in G1 arrested cells and mid-M phase cells. The percentage of cells with 2 GFP spots was plotted. 100–200 cells were scored for each data point and data was generated from two independent experiments. (C) Cohesion loss in smc3 interface mutant mid-M cells. SMC3-AID alone (white), or containing WT (gray), smc3-I1026R (dark blue) and smc3-L1029R (light blue). (D) Cohesion loss in mcd1 mutant mid-M cells. MCD1-AID strain alone (white) or containing, either WT (gray), mcd1-L75K (dark blue) or mcd1-L89K (light blue). (E–F) Protein extracts from the synchronous arrest regimen in A and B were made from G1 cells, auxin treated G1 cells and mid-M cells then subjected to western blot analysis. (E) Mcd1p is degraded in mid-M phase smc3 interface mutants. Top panel detects Smc3p-3V5-AID (αV5), middle panel detects Mcd1p (αMcd1). Tubulin (αTub2p; bottom panel) was used as a loading control. As a control we showed in (Figure 6—figure supplement 2) that the smc3-L1029R mutation did not affect its protein levels by comparing 6 HA-epitope-tagged Smc3p (VG3943-1C) and Smc3p-L1029R (VG3944-3D). (F) Mcd1p interface mutants are degraded in mid-M cells. Top panel detects Mcd1p (αMcd1) with star indicating Mcd1p-AID and arrow head Mcd1p WT and interface mutants, bottom panel detects Tubulin (αTub2p). .