Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset.

We have cloned an unique gene encoding the heavy chain of a type II myosin in the fission yeast, Schizosaccharomyces pombe. The myo2+ gene encodes a protein of 1526 amino acids with a predicted molecular weight of 177 kDa and containing consensus binding motifs for both essential and regulatory light chains. The S. pombe myo2+ head domain is 45% identical to myosin IIs from Saccharomyces cerevisiae and Homo sapiens and 40% identical to Drosophila melanogaster. Structurally, myo2+ most closely resembles budding yeast MYO1, the tails of both myosin IIs containing a number of proline residues that are predicted to substantially disrupt the ability of these myosins to form coiled coils. The myo2+ gene is located on chromosome III, 8.3 map units from ade6+. Deletion of approximately 70% of the coding sequence of myo2+ is lethal but myo2Δ spores can acquire a suppressor mutation that allows them to form viable microcolonies consisting of filaments of branched cells with aberrant septa. Overexpression of myo2+ results in the inhibition of cytokinesis; cells become elongated and multinucleate and fail to assemble a functional cytokinetic actin ring and are either aseptate or form aberrant septa. These results suggest that a contractile actin‐myosin based cytokinetic mechanism appeared early in the evolution of eukaryotic cells and further emphasise the utility of fission yeast as a model organism in which to study the molecular and cellular basis of cytokinesis. Cell Motil. Cytoskeleton 38:385–396, 1997. © 1997 Wiley‐Liss, Inc.

The mechanism for transcriptional silencing of pericentric heterochromatin is conserved from fission yeast to mammals. Silenced genome regions are marked by epigenetic methylation of histone H3, which serves as a binding site for structural heterochromatin proteins. In the fission yeast Schizosaccharomyces pombe, the major structural heterochromatin protein is Swi6. To gain insight into Swi6 function in vivo, we have studied its dynamics in the nucleus of living yeast. We demonstrate that, in contrast to mammalian cells, yeast heterochromatin domains undergo rapid, large-scale motions within the nucleus. Similar to the situation in mammalian cells, Swi6 does not permanently associate with these chromatin domains but binds only transiently to euchromatin and heterochromatin. Swi6 binding dynamics are dependent on growth status and on the silencing factors Clr4 and Rik1, but not Clr1, Clr2, or Clr3. By comparing the kinetics of mutant Swi6 proteins in swi6؊ and swi6 ؉ strains, we demonstrate that homotypic protein-protein interactions via the chromoshadow domain stabilize Swi6 binding to chromatin in vivo. Kinetic modeling allowed quantitative estimation of residence times and indicated the existence of at least two kinetically distinct populations of Swi6 in heterochromatin. The observed dynamics of Swi6 binding are consistent with a stochastic model of heterochromatin and indicate evolutionary conservation of heterochromatin protein binding properties from mammals to yeast.In eukaryotic cells, genomes exist in the form of chromatin. Morphological studies have described two major types of chromatin: euchromatin corresponds to loosely packed chromatin, where most active genes are transcribed, whereas heterochromatin consists of condensed, predominantly transcriptionally repressed chromatin (48). Heterochromatin is molecularly characterized by a high density of nucleosomes containing histone H3 methylated on lysine 9 (H3-K9) (5, 27). In humans, H3-K9 methylation is mediated by methyltransferases Suv39h1 and Suv39h2 (43). The presence of methylated H3-K9 creates a specific binding site for one of the major heterochromatin proteins, HP1 (heterochromatin protein 1) (5,27,37,43). HP1 was originally identified in Drosophila melanogaster as a suppressor of variegation, and it has been shown to modify mammalian position effect variegation in a dose-dependent manner (11,19). The role of HP1 does not appear to be limited to heterochromatin since HP1 also represses, and in some cases activates, euchromatic genes (24, 28). Consistent with its primary role in the formation and maintenance of heterochromatin, HP1 is predominantly localized in heterochromatin domains (12, 33).The system of repression involving methylation of H3-K9 as a mark which is recognized by a structural chromatin protein is conserved from mammalian cells to Schizosaccharomyces pombe. The fission yeast homologues of Suv39h and HP1 are Clr4 and Swi6, respectively (25,30,47). Swi6 binds to three transcriptionally silent heterochromatic regions, the mating ty...

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