A collection of fission yeast Schizosaccharomyces pombe conditional mutants was screened for defective nucleocytoplasmic transport of poly(A)+ RNA by fluorescence in situ hybridization. We identified a temperature-sensitive mutant that accumulated poly(A)+ RNA in the nucleus and have named it rae1-1, for ribonucleic acid export. All rae1-1 cells exhibit the defect in poly(A)+ RNA export within 30 min following a shift to the non-permissive temperature. In addition, in the rae1-1 mutant, actin and tubulin become disorganized, and cells undergo an irreversible cycle arrest. Results from experiments in which rae1-1 cells were arrested in various phases of the cell division cycle and then shifted to nonpermissive temperature suggest that cells are particularly vulnerable to loss of rae1 function during G2/M. However, the inability to export RNA from the nucleus to the cytoplasm was not limited to a particular phase of the cell division cycle. The rae1 gene was isolated by complementation and encodes a predicted protein of 352 amino acids with four beta-transducin/WD40 repeats.

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.

HIV Tat