Bfa1 or Bub2 directly interacts with Tem1 in vitro. To
investigate interactions among recombinant Bfa1, Bub2, and Tem1
proteins under various conditions, partially purified 0.5 μg each of
T7-Bfa1 and T7-Bub2 proteins was added into reaction tubes containing
either bead-bound GST or GST-Tem1, which is supplemented with GDP,
GTP[γS], or GDP + AlF. Proteins associating with
either GST-Tem1 or GST were precipitated and detected by immunoblotting
with an anti-T7 antibody. The same membrane subsequently was blotted
with an anti-GST antibody to detect the precipitated GST and GST-Tem1
fusion protein. Control GST blot was assembled with GST-Tem1 blot from
the same exposure, and the migration difference does not reflect the
actual mobility differences of these two proteins in the gel. Input,
15% of the proteins used in the binding assays; control, GST control
with both Bfa1 and Bub2 in the presence of GDP+AlF.
GDP and GTP[γS] were supplemented at the final concentration of 100
μM, whereas AlF was supplemented at 110 μM.
Bfa1, T7-Bfa1-His-6 recombinant protein; Bub2, T7-Bub2-His-6
recombinant protein.