FIGURE 9.
The Swi6L315E mutant fails to interact with Clr4. A and B, the Swi6L315E mutant fails to interact with Clr4 in vivo. A, inputs. Whole cell extracts from clr4-Myc, the swi6Δ strain transformed with vector alone, GFP-swi6, and the GFP-swi6L315E mutant gene were immunoblotted with α-Myc (upper panel), α-Swi6 (center panel), and α-tubulin antibody as loading control (lower panel). B, samples in A were immunoprecipitated with α-Swi6 antibody, and the immunoprecipitated samples were immunoblotted with α-Myc (upper panel) and α-Swi6 (lower panel) antibodies. The reverse immunoprecipitation reaction was not performed because of poor extractability of myc-tagged Clr4. C, pull-down assay showing interaction of GST-Clr4 with (His)6-tagged Swi6 in vitro. GST (lane 2) and GST-Clr4 (lanes 3–5) were bound to glutathione-Sepharose beads and incubated with increasing concentrations of extracts of E. coli cells expressing (His)6-Swi6 (lanes 2 and 3-5). Lane 1 contains (His)6-Clr4 at 5% input. After washing, the beads were subjected to SDS-PAGE and immunoblotted with α-His antibody. D, far-Western analysis to study the interaction of WT and the Swi6L315E mutant with GST-Clr4. Equal amounts of MBP, MBP-Swi6, and MBP-Swi6L315E proteins were subjected to SDS-PAGE and electroblotted. The membrane was renatured according to Ref. 35 and incubated with purified GST-Clr4 protein, followed by detection using α-GST antibody. E, self-association of Swi6 is independent of Clr4. A glutaraldehyde cross-linking experiment was done with extracts from swi6+, clr4+ (lanes 3–5), and swi6+, clr4− (lanes 8–10) strains. Lanes 3 and 8 represent mock-treated samples, whereas lanes 4, 5, 9, and 10 show the effect of increasing amounts of glutaraldehyde (Gtx). Lanes 1 and 2 represent extracts from swi6−, clr4+ with and without cross-linking, respectively. Similarly, lanes 6 and 7 represent extracts from the swi6−, clr4− strain without and with cross-linking, respectively.