FIG. 3.

Inhibition of transport by GST-M protein occurs from within the nucleus. (A) Stability and distribution of GST-M protein. The nucleocytoplasmic distribution of GST-M protein was monitored by Western blotting with αGST antibody. Extracts were prepared 1, 4, and 20 h after nuclear (top panels) or cytoplasmic (bottom panels) injection of GST-M protein; one oocyte equivalent of the nuclear (N) or cytoplasmic (C) extracts was analyzed. (B) Inhibitory activity of GST-M protein. Import of ³²P-labeled U1, U5, and U6 snRNAs and NL-15 RNA (import RNAs) was analyzed in control oocytes (a) and in oocytes preinjected with GST-M protein in the nucleus (b) or the cytoplasm (c). GST-M protein was injected 2 h prior to injection of import RNAs. Import was monitored 28 h after injection of the RNA mixture (I). (C) Nuclear function of GST-M protein. Export of ³²P-labeled U1_Sm⁻ snRNA and tRNA was analyzed in control oocytes (a) and in oocytes preinjected with GST-M protein (b and d) or with both GST-M protein and αM antibody (c, e, and f). αM was injected 1 h prior to the injection of GST-M protein, which was followed 1 h later by the injection of the export RNAs. Export was monitored 1 h after injection of the RNA mixture (I). (D) Reversal of transport inhibition in the absence of protein synthesis. Export of ³²P-labeled U1_Sm⁻ snRNA and tRNA was analyzed in the presence of cycloheximide in control oocytes (a) and in oocytes preinjected with GST-M protein (b) or with GST-M protein and nuclear αM antibody (c). GST-M protein was injected into the cytoplasm, and 1.5 h later cycloheximide (200 μg/ml) was added to the oocyte incubation medium. αM was injected 1 h after the addition of cycloheximide and 1 h prior to the injection of export RNAs. This amount of cycloheximide was sufficient to block protein synthesis as monitored by [³⁵S]methionine labeling (data not shown).