The fission yeast ran GTPase is required for microtubule integrity.

The microtubule cytoskeleton plays a pivotal role in cytoplasmic organization, cell division, and the correct transmission of genetic information. In a screen designed to identify fission yeast genes required for chromosome segregation, we identified a strain that carries a point mutation in the SpRan GTPase. Ran is an evolutionarily conserved eukaryotic GTPase that directly participates in nucleocytoplasmic transport and whose loss affects many biological processes. Recently a transport-independent effect of Ran on spindle formation in vitro was demonstrated, but the in vivo relevance of these findings was unclear. Here, we report the characterization of a Schizosaccharomyces pombe Ran GTPase partial loss of function mutant in which nucleocytoplasmic protein transport is normal, but the microtubule cytoskeleton is defective, resulting in chromosome missegregation and abnormal cell shape. These abnormalities are exacerbated by microtubule destabilizing drugs, by loss of the spindle checkpoint protein Mph1p, and by mutations in the spindle pole body component Cut11p, indicating that SpRan influences microtubule integrity. As the SpRan mutant phenotype can be partially suppressed by the presence of extra Mal3p, we suggest that SpRan plays a role in microtubule stability.

Mal3, the fission yeast homologue of the human APC-interacting protein EB-1 is required for microtubule integrity and the maintenance of cell form.

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3+. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3+ gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3+ gene product had no gross effect on mitotic spindle morphology, overexpression of mal3+ compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.

A dominant negative allele of p34cdc2 shows altered phosphoamino acid content and sequesters p56cdc13 cyclin.

The cdc2 gene product, a 34-kDa phosphoprotein with serine/threonine protein kinase activity, has been implicated as the key component in the regulation of the eucaryotic cell cycle. Activation of the cdc2 protein kinase is regulated by its phosphorylation state and by interaction with other proteins. We have mutagenized the fission yeast cdc2 gene to obtain conditionally dominant negative alleles. One of these mutants, named DL2, is characterized in this report. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and leads to arrest in the G2 phase of the cell cycle. The mutant phenotype is the result of a single amino acid change in the GDSEID motif of the protein, a region of identity in all cdc2 homologs, and results in a nonfunctional protein that shows an altered content of phosphothreonine. Multicopy suppressors of the dominant negative phenotype have been isolated, and one of these has been shown to encode the cdc13 cyclin B gene product.

Fission yeast mal2+ is required for chromosome segregation.

By a screen designed to isolate new fission yeast genes required for chromosome segregation, we have identified mal2+. The conditionally lethal mal2-1 allele gives rise to increased loss of a nonessential minichromosome at the permissive temperature and leads to severe missegregation of the chromosomes at the nonpermissive temperature. Cloning by complementation and subsequent sequence analysis revealed that mal2 is a novel protein with a mass of 34 kDa. Cells containing a mal2 null allele were inviable, indicating that mal2+ is an essential gene. Fusion of mal2 protein to the green fluorescent protein (GFP) showed that mal2 was predominantly localized in the nucleus. Sensitivity to microtubule-destabilizing drugs and strong genetic interactions with alpha1-tubulin suggest an interaction of the mal2 protein with the microtubule system. Spindle formation and elongation were not detectably affected in the mal2-1 mutant as determined by indirect immunofluorescence. However, anomalous chromosome movement on the spindle leading to aberrant distribution of the chromosomal material was observed.

Construction of LYS2 cartridges for use in genetic manipulations of Saccharomyces cerevisiae.

Linker arrays were added to the 5' and 3' boundaries of the Saccharomyces cerevisiae LYS2 gene, which allow the generation of 18 LYS2 cartridges with different sticky ends. As it was necessary to define the beginning and the end of the approx. 4.5-kb LYS2 gene, we sequenced 1 kb of its 5' and 1.5 kb of its 3' region and mapped the mRNA start point. The open reading frame (ORF) found by this analysis was proven to be the LYS2 ORF by exchanging the sequences upstream from the presumptive ATG with the S. cerevisiae CYC1 promoter and subsequent demonstration of LYS2 expression in vivo. The proper functioning of the LYS2 cartridges was demonstrated by the transformation of lys2 mutant strains to Lys+ prototrophy using plasmids furnished with a LYS2 cartridge.

Expression of a dominant negative allele of cdc2 prevents activation of the endogenous p34cdc2 kinase.

The cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a 34 kDa phosphoprotein with serine/threonine protein kinase activity that acts as the key component in regulation of the eukaryotic cell cycle. We used a repressible promoter fused to the cdc2 cDNA to isolate conditionally dominant negative mutants of cdc2. One of these mutants, DL5, is described in this paper. Overexpression of the mutant protein in a wild-type cdc2 background is lethal and confers cell cycle arrest with a typical cdc- phenotype. Sequencing of the mutant cdc2 gene revealed a single amino acid substitution in a region highly conserved in cdc2-like proteins. The mutant protein exhibits no protein kinase activity, but is able to bind a component(s) required for an active protein kinase complex and thereby prevents binding of this component(s) to the co-existing wild-type cdc2 protein. We also demonstrate that S. pombe p34cdc2 contains no phosphoserine.

The centromere of budding yeast.

Stable maintenance of genetic information during meiosis and mitosis is dependent on accurate chromosome transmission. The centromere is a key component of the segregational machinery that couples chromosomes with the spindle apparatus. Most of what is known about the structure and function of the centromeres has been derived from studies on yeast cells. In Saccharomyces cerevisiae, the centromere DNA requirements for mitotic centromere function have been defined and some of the proteins required for an active complex have been identified. Centromere DNA and the centromere proteins form a complex that has been studied extensively at the chromatin level. Finally, recent findings suggest that assembly and activation of the centromere are integrated in the cell cycle.

Purification and characterization of an auxin-binding protein from Arabidopsis thaliana expressed in baculovirus-infected insect cells.

The auxin-binding protein At-ERabp1 is of very low abundance in Arabidopsis thaliana; it hinders any study at the protein level as it is difficult to collect large amounts from the plant. We therefore chose to express At-ERabp1 in baculovirus-infected insect cells. Recombinant baculoviruses were selected in yeast according to Patel et al. (Nucleic Acids Res. 20, 97-104, 1991). The recombinant protein was purified to homogeneity by a simple procedure involving an affinity step on a succinyl-concanavalin A column. Labeling with the photoactive auxin 5-N3-[7-3H]indole-3-acetic acid demonstrated that the baculovirus-expressed protein belongs to the auxin-binding protein family as deduced from its cDNA homology to a gene previously characterized in maize. The mature polypeptide migrates on SDS-PAGE with an apparent molecular mass of about 23 kDa, its NH2-leader sequence is properly processed, and it bears a high-mannose-type sugar moiety. All results are in agreement with information derived from the cDNA analysis. The possible role of a functional dimerization is also discussed.

Regulation of cdc2 activity in Schizosaccharomyces pombe: the role of phosphorylation.

The cdc2 protein kinase, first identified as a cell cycle gene required for transition into the S- and M-phases of budding and fission yeast, has been shown to act as a key component in the regulation of the eukaryotic cell cycle. The periodic activation of cdc2 kinase, which is required for entry into M-phase, is regulated by subunit association with cyclin B, the cdc25, wee1, mik1 gene products and differential phosphorylation of the cdc2 protein. Phosphorylation at Tyr 15 inhibits activation of the cdc2/cdc13 complex whereas phosphorylation of Thr 167 is required for kinase activity.

Functional selection for the centromere DNA from yeast chromosome VIII.

Centromeres are essential components of eucaryotic chromosomes. In budding yeast, up to now, 15 of the 16 centromere DNAs have been isolated. Here we report the functional isolation and characterization of CEN8, the last of the yeast centromeres missing. The centromere consensus sequence for the 16 chromosomes in this organism is presented.

Complementation of the cs dis2-11 cell cycle mutant of Schizosaccharomyces pombe by a protein phosphatase from Arabidopsis thaliana.

The activities of type I protein phosphatases play a central role in eukaryotic cell cycle control. Here, we report the cloning and characterization from the flowering plant Arabidopsis thaliana of a cDNA clone named PP1-At which is highly homologous to protein phosphatase 1. The deduced amino acid sequence of PP1-At shows that the PP1-At protein is 318 amino acid residues long and has a molecular weight of 35,298 Da. The PP1-At protein has strong similarity to all other known protein phosphatase type 1 catalytic subunits. Approximately 62% of the amino acids are identical to type 1 protein phosphatases of rabbit, mouse, Saccharomyces cerevisiae and Schizosaccharomyces pombe. RNA blot analysis revealed a single mRNA species of approximately the same size as the cDNA isolated. The PP1-At-encoded mRNA of 1.3 kb is abundant in most vegetative Arabidopsis tissues, with the lowest level of expression in leaves. When transferred to the fission yeast S.pombe, the PP1-At-encoded protein can rescue a semidominant mutant, cold sensitive (cs) dis2-11, which under nonpermissive conditions is unable to complete chromosome disjunction.

A phosphorylation site mutant of Schizosaccharomyces pombe cdc2p fails to promote the metaphase to anaphase transition.

The protein kinase cdc2p is a key regulator of the G1-S and G2-M cell cycle transitions in the yeast Schizosaccharomyces pombe. Activation of cdc2p is regulated by its phosphorylation state and by interaction with other proteins. We have analyzed the consequences for cell cycle progression of altering the conserved threonine phosphorylation site, within the activation loop of cdc2p, to glutamic acid. This mutant, T167 E, promotes entry into mitosis, as judged by the accumulation of mitotic spindles and condensed chromosomes, despite the fact that it lacks demonstrable kinase activity both in vitro and in vivo. However, T167 E cannot promote the metaphase-anaphase transition. Since a component of the anaphase-promoting complex (APC) in S. pombe, cut9p, remains hypophosphorylated at the T167 E arrest point, the cell cycle block might be due to the inability of T167 E to activate the APC. T167 E is lethal when overexpressed, and overproduction also causes a mitotic arrest. Multicopy suppressors of the dominant negative phenotype were isolated, and identified as cdc13+ and suc1+. Overexpression of suc1+ suppresses the effects of T167 E overproduction by restoring sufficient amounts of suc1p to the cell to allow passage through mitosis.

A fast method to diagnose chromosome and plasmid loss in Saccharomyces cerevisiae strains.

We have developed a simple, fast and reliable method for the analysis of genetic stability in budding yeast strains. The assay relies on our previous finding that cells expressing the green fluorescent protein (GFP) can be detected and counted by flow cytometric analysis (FACS) (Niedenthal et al., 1996). Expression of a gfp-carrying CEN-plasmid in a wild-type strain resulted in the emission of strong fluorescence from 80% of the cell population. Strong fluorescence and presence of the plasmid, determined by the presence of the URA3 genetic marker, was strictly correlated. Expression of this plasmid in 266 yeast strains, each carrying a complete deletion of a novel, non-essential gene identified in the S. cerevisiae sequencing project, pinpointed 12 strains with an increased level of mitotic plasmid loss. Finally we have shown that measurement of mitotic loss of artificial chromosome fragments equipped with the gfp expression cassette can be performed quantitatively using FACS.

All 16 centromere DNAs from Saccharomyces cerevisiae show DNA curvature.

All 16 centromere DNA regions of Saccharomyces cerevisiae including 90 bp framing sequences on either side were cloned. These 300 bp long centromere regions were analysed by native polyacrylamide gel electrophoresis and found to display a reduced mobility indicative of DNA curvature. The degree of curvature is centromere dependent. The experimental data were confirmed by computer analysis of the 3-dimensional structure of the CEN DNAs. Altogether these data provide further evidence for a model for budding yeast centromeres in which CEN DNA structure could be important for the assembly, activity and/or regulation of the centromere protein-DNA complex.

The sequence of a 24,152 bp segment from the left arm of chromosome XIV from Saccharomyces cerevisiae between the BNI1 and the POL2 genes.

In the framework of the European Union programme for sequencing the genome of Saccharomyces cerevisiae we have determined the nucleotide sequence of a region of 24,152 bp located on the left arm of chromosome XIV between the BNI1 and the POL2 genes. The sequence was obtained by directed sequence analysis using a mixture of ExoIII and primer walking strategies. Subsequent analysis revealed 13 open reading frames (ORFs) including four small ORFs completely internal to, or partly overlapping with, other ORFs. Five of these ORFs have been described previously (BNI1, APL1, LYP1, PIK1, POL2) and thus 74.8% of the 24,152 bp were already present in the databases prior to this sequencing effort. Interestingly, all 13 identified ORFs are characterized by a low codon adaptation index (0.04-0.22). In addition, this region of chromosome XIV shows an unusually high gene density with about 88% of coding DNA. This amounts to one gene per 2177 bp, which is significantly above the average gene length (about 1500 bp). For eight ORFs considerable homologies to 'Expressed Sequence Tags' derived from human cDNAs located in the XREF database could be identified.

The vesicular transport protein Cgp1p/Vps54p/Tcs3p/Luv1p is required for the integrity of the actin cytoskeleton.

The CGP1 gene was identified in a screen for mutations that were synthetic lethal in combination with a deletion of the gene (CPF1) for centromere and promoter factor 1. Cells deleted for CGP1 showed reduced viability, were temperature sensitive for growth and exhibited altered sensitivity to microtubule-destabilizing drugs. Furthermore, Deltacgp1 cells showed increased rates of loss of a circular minichromosome and defects in the positioning of the short mitotic spindle. Further phenotypic analysis of Deltacgp1 cells revealed that loss of Cgp1p function led to severe depolarization of the actin cytoskeleton. In addition, cells deleted for CGP1 were hypersensitive to the actin-disrupting compound Latrunculin-A, exhibited strongly reduced polarized localization of the unconventional myosin Myo2p, and showed defects in other actin-related processes, such as shmoo formation and cell wall integrity. Cgp1p was recently identified by several groups as Vps54p, which is a member of the VFT complex that is involved in vesicular protein transport at the level of the late Golgi, acting as a tethering factor. Our data show for the first time that Cgp1p/Vps54p links aspects of vesicular protein transport with the organization of the actin cytoskeleton.

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.