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Fig. 4. Import of GFP-SRP19 into the germinal vesicle and export back to the cytoplasm. Samples of GFP-SRP19 (20 ng) were injected into the cytoplasm of oocytes, and GVs and cytoplasms were isolated under oil at 1, 2, 5 and 10 hours (h) after injection. (A) Protein was extracted and analysed by immunoblotting using antibodies to GFP. Bands at 47 kDa corresponding to the mass of the fusion protein (lane S, noninjected protein sample) show a steady increase in translocation from cytoplasm (C) to GV (N). (B) Ratios of nuclear to cytoplasmic concentration ([N]/[C]) were calculated from band density scans and mean values from four different injection experiments were plotted against time (open circles). In a separate series of experiments, co-injection of 25 ng of the import receptor importin- shows an enhanced initial rate of nuclear uptake of GFP-SRP19 (filled circles). (C) Export of GFP-SRP19 from the germinal vesicle to the cytoplasm. From 10 hours after injection of GFP-SRP19 into the cytoplasm the nuclear to cytoplasmic concentration ratio ([N]/[C]) starts to fall (white columns), indicating export from the GV. This fall is inhibited by incubating the oocytes in the presence of 50 nM leptomycin B (LMB, grey columns).

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