Figure 5.

(A-C) Cooperative binding of NES, Yrb1p, and Yrb2p to Xpo1p/Gsp1p-GTP. Gsp1p[γ-³²P]GTP (50 pM) was incubated with 400 nM Xpo1p and the indicated concentrations of NES peptides or proteins (A and B) or Yrb1p or Yrb2p (C). Reactions containing Gsp1p[γ-³²P]GTP and Yrb1p or Yrb2p alone served as controls. After 30 min, the GTPase reactions were started by addition of 40 nM Rna1p. Hydrolysis of GTP by Gsp1p was determined as released [³²P]phosphate after 30 s by the charcoal method. (D) Kinetics of Rna1p-induced complex disassembly. Gsp1p[γ-³²P]GTP (50 pM) was incubated for 30 min with 500 nM Xpo1p alone, with Xpo1p and 100 nM Yrb1p, or with Xpo1p and 100 nM Yrb2p. After Rna1p addition, the reactions were incubated further for the indicated time periods. GTP hydrolysis was then determined by the charcoal method.