Fig 3.

Rtt109(1–424) physically interacts with Vps75 in vivo and can acetylate H3K9 in the presence of Vps75 in vitro. (A) 12Myc-Rtt109(1–424) coimmunoprecipitates with Vps75-TAP no differently than 12Myc-Rtt109. Vps75-TAP was immunoprecipitated from the indicated strains with IgG-Sepharose beads as described in Materials and Methods. WCE and IPs were resolved by 12% SDS-PAGE, transferred onto nitrocellulose paper, and immunoblotted with anti-Myc and anti-TAP. (B) Rtt109(1–424)-Vps75 is able to acetylate H3K9 in vitro. HAT assays were performed using indicated recombinant proteins with acetyl-CoA and chicken core histones for 30 min, resolved by 10% and 15% SDS-PAGE, and transferred onto nitrocellulose. The 10% gel was immunoblotted with anti-6×HIS, whereas the 15% gel was immunoblotted with anti-H3K56ac and anti-H3K9ac. The asterisk indicates a breakdown product of 6×HIS-Vps75. (C) Rtt109(1–424)-Vps75 catalyzes in vitro H3K9ac at the same efficiency as Rtt109-Vps75. Serial dilutions of equal amounts of 6×HIS-Rtt109 and 6×HIS-Rtt109(1–424) were incubated with a constant amount of 6×HIS-Vps75. HAT reactions were carried out as described for panel B using as substrate a mixture of core and linker histones purified from chicken erythrocytes. Reactions were resolved by 15% SDS-PAGE and Coomassie stained or transferred onto nitrocellulose and immunoblotted as described for panel B. Lane M, molecular mass marker.