Figure 3.

S Compartments: Coiled Coils, E Head, and J Head Interactions in Cohesin Heterodimers

(A) E state of Smc heads in heterodimers. Smc1 and Smc3-HaloTag proteins containing hinge and/or E heads cysteine pairs were cross-linked in vivo using BMOE. Complexes were immunoprecipitated on Scc1-PK6, labeled with TMR ligand, separated by SDS-PAGE, and quantified by in-gel fluorescence. Percentage (mean ± SD of 3 independent experiments) of cross-linking efficiency is given. Asterisk shows the location of the double cross-link. ICC1 = 0.74.

(B) E Smc heads and coiled coils interactions in heterodimers. Smc1 and Smc3-HaloTag proteins containing hinge, E heads, and/or coiled coils cysteine pairs were analyzed as in (A). ICC1 = 0.84.

(C) J state of Smc heads in heterodimers. Smc1-HA, Smc3, and Scc1-PK proteins containing hinge and/or J heads cysteine pairs were cross-linked in vivo, immunoprecipitated on Scc1-PK6, separated by SDS-PAGE, and analyzed by western blot. ICC1 = 0.37.

(D) J heads interactions with Smc coiled coils. Smc1 and Smc3-HaloTag proteins containing coiled coils and/or alternative J heads cysteine pairs were analyzed as in (A). ICC1 = 0.84.

(E) E and J states of Smc heads are mutually exclusive. Smc1 and Smc3-HaloTag proteins containing E heads and/or J heads cysteine pairs were analyzed as in (A). Percentage of the double cross-link efficiency is shown in box. Left lane: double cross-link of E heads with coiled coils shown for size indication. ICC1 = 0.52.

See also Figure S2.