Fission yeast temperature‐sensitive mutants cut3‐477 and cut14‐208 fail to condense chromosomes but small portions of the chromosomes can separate along the spindle during mitosis, producing phi‐shaped chromosomes. Septation and cell division occur in the absence of normal nuclear division, causing the cut phenotype. Fluorescence in situ hybridization demonstrated that the contraction of the chromosome arm during mitosis was defective. Mutant chromosomes are apparently not rigid enough to be transported poleward by the spindle. Loss of the cut3 protein by gene disruption fails to maintain the nuclear chromatin architecture even in interphase. Both cut3 and cut14 proteins contain a putative nucleoside triphosphate (NTP)‐binding domain and belong to the same ubiquitous protein family which includes the budding yeast Smc1 protein. The cut3 mutant was suppressed by an increase in the cut14+ gene dosage. The cut3 protein, having the highest similarity to the mouse protein, is localized in the nucleus throughout the cell cycle. Plasmids carrying the DNA topoisomerase I gene partly suppressed the temperature sensitive phenotype of cut3‐477, suggesting that the cut3 protein might be involved in chromosome DNA topology.

Nutrients are essential for cell growth and division. Screening of Schizosaccharomyces pombe temperature-sensitive strains led to the isolation of a nutrient-insensitive mutant, tor2-287 . This mutant produces a nitrogen starvation-induced arrest phenotype in rich media, fails to recover from the arrest, and is hypersensitive to rapamycin. The L2048S substitution mutation in the catalytic domain in close proximity to the adenine base of ATP is unique as it is the sole known genetic cause of rapamycin hypersensitivity. Localization of Tor2 was speckled in the vegetative cytoplasm, and both speckled and membranous in the arrested cell cytoplasm. Using mass spectroscopic analysis, we identified six subunits (Tco89, Bit61, Toc1, Tel2, Tti1 and Cka1) that, in addition to the six previously identified subunits (Tor1, Tor2, Mip1/Raptor, Ste20/Rictor, Sin1/ Avo1 and Wat1/Lst8), comprise the TOR complexes (TORCs). All of the subunits so far examined are multiply phosphorylated. Tel2 bound to Tti1 interacts with various phosphatidyl inositol kinase (PIK)-related kinases including Tra1, Tra2 and Rad3, as well as Tor1 and Tor2. Schizosaccharomyces pombe TORCs should thus be functionally redundant and might be broadly regulated through different subunits that are either common or specific to the two TORCs, or even common to various PIK-related kinases. Functional redundancy of the TORCs may explain the rapamycin hypersensitivity of tor2-287 .

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The 20S cyclosome complex (also known as the anaphase-promoting complex) has ubiquitin ligase activity and is required for mitotic cyclin destruction and sister chromatid separation. The formation and activation of the 20S cyclosome complex is regulated by an unknown mechanism. Here we show that Cut4 (ref. 6) is an essential component of the cyclosome in fission yeast. Cut4 shares sequence similarity with BimE, a protein that regulates mitosis in Aspergillus nidulans. Mutations in cut4 result in hypersensitivity to cyclic AMP and to stress-inducing heavy metals, inhibition of the onset of anaphase, disruption of the 20S complex, and inhibition of mitotic cyclin ubiquitination. These phenotypes are fully suppressed by cAMP phosphodiesterase and the protein kinase A (PKA) regulatory subunit and weakly suppressed by Sti1 (an activator of the Hsp70 and Hsp90 chaperones). Suppression correlates with the amount of 20S complex, indicating that cyclosome formation and activation is inhibited by the cAMP/PKA pathway.

Fission yeast cold-sensitive (cs) disl mutants are defective in sister chromatid separation. The dis1 + gene was isolated by chromosome walking. The null mutant showed the same phenotype as that of cs mutants. The d/s/+ gene product was identified as a novel 93-kD protein, and its localization was determined by use of anti-disl antibodies and green fluorescent protein (GFP) tagged to the carboxyl end of p93 dis1. The tagged p93 dlsl in living cells localizes along cytoplasmic microtubule arrays in interphase and the elongating anaphase spindle in mitosis, but association with the short metaphase spindle microtubules is strikingly reduced. In the spindle, the tagged p93 ais~ is enriched at the spindle pole bodies (SPBs). Time-lapse video images of single cells support the localization shift of p93 dis~ to the SPBs in metaphase and spindle microtubules in anaphase. The carboxy-terminal fragment, which is essential for Disl function, accumulates around the mitotic SPB. We propose that these localization shifts of p93 dis~ in mitosis facilitates sister chromatid separation by affecting SPB and anaphase spindle function.