No abstract

Cyclins, as subunits of the protein kinase encoded by the cdc2 gene are major controlling elements of the eukaryotic cell cycle. The fission yeast Schizosaccharomyces pombe has a B-type cyclin, which is a nuclear protein encoded by the cdc13 gene. Here we demonstrate the presence of two spatially distinct cdc13 cyclin populations in the nucleus of S. pombe, one of which is associated with the mitotic spindle poles. Both populations colocalize with the product of the cdc2 gene (p34cdc2). Treatment of cells with the antimicrotubule drug thiabendazole prevents cyclin degradation and blocks the tyrosine dephosphorylation and activation of cdc2. These results suggest a key regulatory role of the cdc2-cyclin complex in the initiation of mitotic spindle formation and also that mitotic microtubule function is required for cdc2 activation.

SummaryEntry into mitosis in the fission yeast Schizosaccharomyces pombe involves the interaction of a number of genes with the major cell cycle control gene, cdc2+. One of these, cd cl3 +, encodes a protein with homology to cyclin. By indirect immunofluorescence microscopy using antibodies to the appropriate bacterially-expressed protein, we have shown that both cdcl3 and cdc2 are nuclear proteins in S. pombe. Both are localised to a nuclear domain distinct from that occupied by the DAPI-staining chromatin. The immunofluorescence signals of both proteins show a progressive increase during interphase but are undetectable at mitosis. Loss of cdcl3 fluorescence at mitosis reflects the destruction of the protein. Thus, it behaves as a classic cyclin. This is not the case for cdc2, the level of which remains constant through the cell cycle. Cells carrying a disrupted copy of the c d cl3 + gene fail to accumulate either cdcl3 or cdc2 in the nucleus. Cells carrying a disrupted cdc2+ gene fail to accumulate cdc2 but reveal apparently normal levels of cdcl3. cdcl3 therefore appears to be required to localise cdc2 to the nucleus but not vice versa. The destruction of cdcl3 at mitosis may allow cdc2 to redistribute to the cytoplasm.

No abstract

The state of tubulin tyrosination in the fission yeast Schizosaccharomyces pombe was investigated using a combination of indirect immunofluorescence microscopy and Western blotting. Antibodies specific for the tyrosinated form of alpha-tubulin stained all microtubule arrays in wild type cells and recognised the two alpha-tubulin polypeptides in Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography. Antisera that specifically recognised the detyrosinated, glu, form, on the other hand, gave consistently negative results, both in cells undergoing rapid exponential growth and in those allowed to accumulate in stationary phase. Neither the "ageing" of microtubules, by arresting cells at different points (late G1 or G2/M) in the cell division cycle, nor stabilising them, using D2O, lead to any detectable tubulin detryrosination. These results suggest that S. pombe lacks the carboxypeptidase that carries out the tubulin detyrosination reaction. This is the first report of an organism that possesses the correct C-terminal alpha-tubulin sequence yet fails to carry out this post-translational modification. The implication of this novel finding for the biological role of these events is discussed.