Mitsuhiro Yanagida

CRM1 is responsible for intracellular transport mediated by the nuclear export signal

The discovery of nuclear export signals (NESs) in a number of proteins revealed the occurrence of signal-dependent transport of proteins from the nucleus to the cytoplasm. Although the consensus motif of the NESs has been shown to be a leucine-rich, short amino-acid sequence,,, its receptor has not been identified. A cytotoxin leptomycin B (LMB) has recently been suggested to inhibit the NES-mediated transport of Rev protein. Here we show that LMB is a potent and specific inhibitor of the NES-dependent nuclear export of proteins. Moreover, we have found a protein of relative molecular mass 110K (p110) in Xenopus oocyte extracts that binds to the intact NES but not to the mutated, non-functional NES. The binding of p110 to NES is inhibited by LMB. We show that p110 is CRM1, which is an evolutionarily conserved protein originally found as an essential nuclear protein in fission yeast and known as a likely target of LMB. We also show that nuclear export of a fission yeast protein, Dsk1, which has a leucine-rich NES, is disrupted in wild-type yeast treated with LMB or in the crm1 mutant. These results indicate that CRM1 is an essential mediator of the NES-dependent nuclear export of proteins in eukaryotic cells.

The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases

S. pombe dis mutants block mitotic chromosome disjunction in a manner reminiscent of aneuploidy formation, and belong to three distinct genes, dis1-dis3. We cloned two independent genomic DNAs that complemented both the cold-sensitive and caffeine-hypersensitive phenotype of dis2-11. These genes, dis2+ and a suppressor sds21+, encode proteins (calculated MW 37,000) with similar predicted amino acid sequences. dis2+ and sds21+ have overlapping functions, and disruptants are lethal only when both genes are disrupted. The gene products identified by anti-dis2 serum are enriched in nuclei. By hybridization, we obtained two cDNA clones from mouse and one genomic clone from S. cerevisiae; the latter complements S. pombe dis2-11. These dis2+ and similar polypeptides of yeasts and mouse are found to be highly homologous (75%-90% identical) to rabbit protein phosphatase 1. The implications of these findings are discussed with regard to mitotic control.

The NDA3 gene of fission yeast encodes β-tubulin: A cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis

The cells of a cold-sensitive mutant nda3-KM311 of the fission yeast Schizosaccharomyces pombe were arrested highly synchronously at a step similar to mitotic prophase when incubated at a restrictive temperature. DAPI staining and indirect immunofluorescence microscopy showed three condensed chromosomes but no spindle. Six minutes after the temperature shifted to a permissive one, the spindle appeared and elongated. The chromosomes were separated at a constant speed (relative velocity 1 micron/min), and the spindle disappeared after the chromosomes reached opposite ends of the cell. The NDA3 gene of S. pombe was cloned by transformation. The 2.6 kb Hind III genomic DNA that complemented the nda3 mutations had only one coding frame split with five short introns. The predicted amino acid sequence contained 448 residues, and was 75% homologous to that of chicken beta-tubulin.

Mis16 and Mis18 Are Required for CENP-A Loading and Histone Deacetylation at Centromeres

Centromeres contain specialized chromatin that includes the centromere-specific histone H3 variant, spCENP-A/Cnp1. Here we report identification of five fission yeast centromere proteins, Mis14-18. Mis14 is recruited to kinetochores independently of CENP-A, and, conversely, CENP-A does not require Mis14 to associate with centromeres. In contrast, Mis15, Mis16 (strong similarity with human RbAp48 and RbAp46), Mis17, and Mis18 are all part of the CENP-A recruitment pathway. Mis15 and Mis17 form an evolutionarily conserved complex that also includes Mis6. Mis16 and Mis18 form a complex and maintain the deacetylated state of histones specifically in the central core of centromeres. Mis16 and Mis18 are the most upstream factors in kinetochore assembly as they can associate with kinetochores in all kinetochore mutants except for mis18 and mis16, respectively. RNAi knockdown in human cells shows that Mis16 function is conserved as RbAp48 and RbAp46 are both required for localization of human CENP-A.

Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization.

We have isolated mutants defective in DNA topoisomerases and an endonuclease from the fission yeast Schizosaccharomyces pombe by screening individual extracts of mutagenized cells. Two type I topoisomerase mutants (top1) and three endonuclease mutants (end1) were all viable. The double mutant top1 end1 was also viable and, in its extract, Mg2+‐ and ATP‐ dependent type II activity could be detected. Three temperature‐sensitive (ts‐) mutants having heat‐sensitive (hs‐) type II enzymes were isolated, and the ts‐ marker cosegregated with the hs‐ type II activity. All the ts‐ mutations fell in one gene (top2) tightly linked to leul in chromosome II. The nuclear division of single top2 mutants was blocked at the restrictive temperature, but the formation of a septum was not inhibited so that the nucleus was cut across with the cell plate. In contrast, the double top1 top2 mutants were rapidly arrested at various stages of the cell cycle, showing a strikingly altered nuclear chromatin region. The type II topoisomerase may have an essential role in the compaction and/or segregation of chromosomes during the nuclear division but also complement the defect of the type I enzyme whose major function is the maintenance of chromatin organization throughout the cell cycle.

Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis.

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.

Establishing Biorientation Occurs with Precocious Separation of the Sister Kinetochores, but Not the Arms, in the Early Spindle of Budding Yeast

Sister kinetochores are bioriented toward the spindle poles in higher eukaryotic prometaphase before chromosome segregation. We show that, in budding yeast, the sister kinetochores are separated in the very early spindle, while the sister arms remain associated. Biorientation of the separated kinetochores is achieved already after replication. Mtw1p, a homolog of fission yeast Mis12 required for biorientation, locates at the centromeres in an Ndc10p-dependent manner. Mtw1p and the sequences 1.8 and 3.8 kb from CEN3 and CEN15, respectively, behave like the precociously separated kinetochores, whereas the sequences 23 and 35 kb distant from CEN3 and CEN5 previously used as the centromere markers behave like a part of the arm. Mtw1p and Ndc10p are identically located except for additional spindle localization of Ndc10p. A model explaining small centromeres and early spindle formation in budding yeast is proposed.

Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle

The activities of type 1 protein phosphatase (PP1) and 2A (PP2A) have distinct, essential roles in cell cycle control. Two previously identified PP1 genes (dis2+ and sds21+) and two PP2A genes (ppa1+ and ppa2+), highly homologous to mammalian PP2A, have been isolated from fission yeast. Only double gene disruption of both PP2A genes results in lethality, as is the case for PP1 genes. By fractionating and assaying PPases in wild-type, various deletion, and point mutant strains, the decrease of PP1 or PP2A activity is shown to cause mitotic defects, exhibiting strikingly different cell cycle phenotypes: cold-sensitive mutations in the same amino acid lesion of PP1 and PP2A produce chromosome nondisjunction and premature mitosis, respectively. Consistently, PP1 and PP2A genes cannot be functionally substituted. Although the overall levels of PP1 and PP2A activities do not fluctuate during the cell cycle, subpopulations might be regulated.

A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1

Defects in kinetochore proteins often lead to aneuploidy and cancer. Mis12–Mtw1 is a conserved, essential kinetochore protein family. Here, we show that a Mis12 core complex exists in Schizosaccharomyces pombe and human cells. Nine polypeptides bind to human hMis12; two of these, HEC1 and Zwint-1, are authentic kinetochore proteins. Four other human proteins of unknown function (c20orf172, DC8, PMF1 and KIAA1570) correspond to yeast Mis12–Mtw1 complex components and are shown to be required for chromosome segregation in HeLa cells using RNA interference (RNAi). Surprisingly, hMis12 also forms a stable complex with the centromeric heterochromatin components HP1α and HP1γ. Double HP1 RNAi abolishes kinetochore localization of hMis12 and DC8. Therefore, centromeric HP1 may be the base to anchor the hMis12 core complex that is enriched with coiled coils and extends to outer Zwint-1 during mitosis.

Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis

By examining cytological phenotypes of 587 temperature‐sensitive mutants of the fission yeast Schizosaccharomyces pombe, we obtained 18 mutants which cause cell division in the absence of nuclear division. By genetic analyses, these novel nuclear division arrest mutants can be classified into nine complementation groups (designated cut1 – cut9). The cytological phenotype of cut mutants is similar but not identical to that of DNA topoisomerase II mutants (top2). The cut1+ gene was cloned by transformation and shown to complement cut2 as well as cut1, indicating a functional relationship between the two genes. The cut genes are required for nuclear division, but their mutant phenotypes differ from most of the previously identified mutants which block nuclear division and also the subsequent cytokinesis. Fluorescence microscopy indicates that the mitotic chromosomes formed in cut mutant cells are abnormal and fail to separate properly. We suggest that cut mutations, like top2, block mitotic chromosome formation and concomitantly nuclear division, but that cytokinesis proceeds independently of the defects in nuclear division, demonstrating uncoordinated mitotic pathways. A novel mutant nuc1 is also described which shows a cytological phenotype similar to the double mutant of DNA topoisomerases I and II but contains normal levels of both DNA topoisomerase activities.