FIGURE 4.

Mtr4-20p and Trf4p are in a complex. Tap-tagged Mtr4p (Y358) and Mtr4-20p (Y359) were purified from whole cell extracts (Materials and Methods). Equivalent amounts of affinity-purified Mtr4p, as determined by immunoblot using anti-Mtr4p polyclonal serum, and whole cell extracts (40 μg) were separated by SDS-PAGE and transferred to a membrane for immunoblot. Rabbit anti-Trf4p or anti-Mtr4p polyclonal serums were used to probe for the respective proteins followed by hrp-coupled goat anti-rabbit IgG antibodies and ECL for detection. Mtr4p with altered mobility due to the presence of the Tap-tag is indicated with an asterisk. (Lane 1) trf4Δ (F22), (lanes 2,3) purified Mtr4p complexes from Tap-Mtr4 (Y358) and Tap-mtr4-20, and (lane 4) the equivalent amount of protein found in lanes 2 and 3 except purified using extract containing untagged Mtr4p (Y200).