The Srp1p/Cse1p/Gsp1p-GTP complex is
sensitive to Yrb1p but resistant to Yrb1p complexed to Gsp1p-GTP.
GST-Srp1p (8 μg per reaction) was immobilized on glutathione
Sepharose and incubated with 12 μg of Cse1p and 4 μg of
Gsp1pQ71L-GTP for 30 min at 4°C. After three washes, bound material
was incubated with buffer alone (lane 5), with 5 μg of Yrb1p (lane
6), with a preincubated mixture of Yrb1p and Gsp1pQ71L-GTP (lane 7), or
with 10 μg of Gsp1pQ71L-GTP (lane 8). After further incubation for 30
min at 4°C, bound (lanes 5–8) and unbound (lanes 9–12) material was
separated by SDS PAGE and analyzed by Coomassie blue staining. The
molecular weight markers (M) and protein loads are shown in lanes
1–4.