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Fig. 6. Interaction of GST-V with importin- in vitro is influenced by the presence of C-terminal phosphorylation sites. (A) Binding of endogenous importin- from an oocyte SN100 fraction to GST-V, pre-phosphorylated GST-V (Vp), GST-V with deletion of the C-terminal 5 kDa (V-5) and GST, all immobilized on glutathione-Sepharose. Immunoblot using anti-importin . (B) Elution of importin- from immobilized GST-V in the presence and absence of Xenopus GV extract, 1 mM ATP and DRB. Importin- was first bound as in A, track 2. (C) Dependence of release of importin- from immobilized GST-V by addition of ATP (filled circles) rather than GTP (open circles). As in B, track 3, but with different concentrations of ATP or GTP. Immunoblots were scanned and relative intensities of signal from the importin- bands were calculated and plotted as a percentage of input protein released. (D) Binding of pre-phosphorylated and non-phosphorylated GST-V, 3m, T445A, 4m and of GST to importin- immobilized on nitrocellulose filters. Immunoblot using anti-GST. (E) Release of pre-phosphorylated and non-phosphorylated GST-V and 3m from immobilized importin- after treatment of filters with GV extract plus ATP. Protein eluted (E) and retained (R) was detected using anti-GST.

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