The C13Y and K56R mutations abolish the scaffolding function of Rad4 <i>in vitro</i>.

Abstract

<p><b>A</b>, schematic diagram of the Rad4 fragments used in this experiment. Each fragment was fused with a GST tag for protein purification and detection in the binding assay using anti-GST antibodies. <b>B</b>, preferential binding of the N- and C-terminal pair of BRCT repeats of Rad4 to Crb2 and Rad9, respectively. Recombinant GST or GST-tagged Rad4 fragments were incubated with immunopurified Crb2 (middle panels) or Rad9 (right panels) bound to the anti-HA antibody beads. The bound proteins were released from the beads in gel loading buffer and analyzed by Western blotting. The membrane was striped and reprobed with anti-HA antibody for Crb2 and Rad9 (bottom panels). Aliquots of the binding reaction were analyzed separately by SDS PAGE as the input (left panel). <b>C</b>, equal amount of the Rad4 fragment b with or without the C13Y-K56R mutation was incubated with Crb2 bound on beads. The input and the Rad4 protein bound to Crb2 were analyzed as in B. <b>D</b>, fragment e with or without the E368K mutation was similarly tested for binding to phosphorylated Rad9. Phosphorylation of Rad9-Thr⁴¹² was detected by blotting of the same membrane with the phospho-specific antibody.</p