Dynein promotes achiasmate segregation in Schizosaccharomyces pombe

Most organisms use crossovers (chiasmata) to maintain physical connections between homologous chromosomes that ensure their proper segregation at the first meiotic division. The fission yeast Schizosaccharomyces pombe has a residual ability to segregate homologous chromosomes in the absence of meiotic recombination (achiasmate segregation). Using cytologically tagged chromosomes, we established a role for the microtubule motor dynein in meiotic chromosome segregation. Dhc1, the motor subunit of dynein, is required for chromosome segregation in both the presence and the absence of recombination. Dlc1, a member of the Tctex-1 dynein light-chain family, preferentially affects the segregation of achiasmate chromosomes. Dlc1 is the first identified protein, outside of Drosophila, that preferentially affects achiasmate chromosome segregation. We discuss possible roles of the dynein motor in this process.     

The 5S RNA genes of Schizosaccharomyces pombe.

The genomic arrangement and sequences of S. pombe 5S RNA genes are reported here. The 5S gene sequences appear to be dispersed within the genome, and are found independently of other rRNA genes. The sequences of two 5S genes examined show identical coding regions of 119 base pairs but have widely varying flanking sequences. A tRNAAsp gene is found in the 3' flanking region of one of the 5S genes. The tRNAAsp gene is faithfully transcribed in an X. laevis in vitro system, while the 5S genes are not transcribed in this system. The phylogenetic position of S. pombe is examined through comparison of 5S RNA sequences.     

Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae

Summary The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNA _Ser ^UGA tRNA gene is followed by a sequence coding for the initiator tRNA^Met. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.     

Malate transport in Schizosaccharomyces pombe.

The transport of malate was studied in a Schizosaccharomyces pombe wild-type strain and in mutant strains unable to utilize malic acid. Two groups of such mutants, i.e., malic enzyme-deficient and malate transport-defective mutants, were differentiated by a 14C-labeled L-malate transport assay and by starch gel electrophoresis followed by activity staining for malic enzyme (malate dehydrogenase [oxaloacetate decarboxylating] [NAD+]; 1.1.1.38) and malate dehydrogenase (1.1.1.37). Transport of malate in S. pombe was constitutive and strongly inhibited by inhibitors of oxidative phosphorylation and of the formulation of proton gradients. Transport was a saturable function of the malate concentration. The apparent Km and Vmax values for transport by the parent were 3.7 mM and 40 nmol/min per mg of protein, respectively, while those of the malic enzyme-deficient mutant were 5.7 mM and 33 nmol/min per mg of protein, respectively. Malate transport was pH and temperature dependent. The specificity of transport was studied with various substrates, including mono- and dicarboxylic acids, and the possibility of a common transport system for dicarboxylic acids is discussed.     

Nif1, a novel mitotic inhibitor in Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, the activity of the M-phase-inducing Cdc2/Cdc13 cyclin-dependent kinase is inhibited by Wee1 and Mik1 tyrosine kinases, and activated by Cdc25 and Pyp3 tyrosine phosphatases. Cdc2/Cdc13 activity is also indirectly regulated by the approximately 70 kDa Nim1 (Cdrl) serine/threonine kinase, which promotes mitosis by inhibiting Wee1 via direct phosphorylation. To understand better the function and regulation of Nim1, the yeast two-hybrid system was used to isolate S.pombe cDNA clones encoding proteins that interact with Nim1. Sixteen of the 17 cDNA clones were derived from the same gene, named nif1 + (nim1 interacting factor-1). Nif1 is a novel approximately 75 kDa protein containing a leucine zipper motif. The Nif1-Nim1 interaction requires a small region of Nim1 that immediately follows the N-terminal catalytic domain. This region is required for Nim1 activity both in vivo and in vitro. delta nif1 mutants are approximately 10% smaller than wild type, indicating that Nif1 is involved in inhibiting the onset of mitosis. Consistent with this proposal, overproduction of Nif1 was found to cause a cell elongation phenotype that is very similar to delta nim1 mutants. Nif1 overproduction causes cell cycle arrest in cells that are partly defective for Cdc25 activity, but has no effect in delta nim1 or delta wee1 mutants. Nif1 also inhibits Nim1-mediated phosphorylation of Wee1 in an insect cell expression system. These observations strongly suggest that Nif1 negatively regulates the onset of mitosis by a novel mechanism, namely inhibiting Nim1 kinase.     

Protein prenylation in Schizosaccharomyces pombe.

S. pombe is shown to be a powerful system for studies concerning attachment of polyisoprenoid moieties to proteins, due to its ability to take up exogenous mevalonic acid efficiently. The fission yeast can take up about 5% of the exogenously added mevalonic acid and incorporate approximately 10% of this into protein. By contrast, the uptake obtained with the budding yeast S. cerevisiae is less than 0.5%. HPLC analysis of total S. pombe protein-bound isoprenoids revealed that approximately 55% of the counts co-migrated with the geranylgeraniol standard, while approximately 45% of the counts co-migrated with farnesol. We could not detect any effects of mevinolin or other HMG-CoA reductase inhibitors in S. pombe.     

The 26S proteasome of the fission yeast Schizosaccharomyces pombe

The 26S proteasome is the multiprotein complex that degrades proteins that have been marked for destruction by the ubiquitin pathway. It is made up of two multisubunit complexes, the 20S catalytic core and the 19S regulatory complex. We describe the isolation and characterization of conditional mutants in the regulatory complex and their use to investigate interactions between different subunits. In addition we have investigated the localization of the 26S proteasome in fission yeast, by immunofluorescence in fixed cells and live cells with the use of a GFP-tagged subunit. Surprisingly, we find that in mitotic cells the 26S proteasome occupies a discrete intracellular compartment, the nuclear periphery. Electron microscopic analysis demonstrates that the complex resides inside the nuclear envelope. During meiosis the localization showed a more dynamic distribution. In meiosis I the proteasome remained around the nuclear periphery. However, during meiosis II there was a dramatic relocalization: initially, the signal occupied the area between the dividing nuclei, but at the end of mitosis the signal dispersed, returning to the nuclear periphery on ascospore formation. This observation implies that the nuclear periphery is a major site of proteolysis in yeast during mitotic growth and raises important questions about the function of the 26S proteasome in protein degradation.     

Cloning and regulation of Schizosaccharomyces pombe thi2, a gene involved in thiamine biosynthesis.

Biosyntheses of the pyrimidine and thiazole moieties of the thiamine molecule occur by separate pathways. In Schizosaccharomyces pombe, a gene, thi2, is responsible for thiazole synthesis [Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We have cloned a 3.1-kb genomic S. pombe fragment which can functionally complement a thi2 mutant. The fragment maps genetically at the thi2 site, indicating that it carries thi2. As shown by Northern hybridization analysis, the appearance of thi2 mRNA levels is repressed when cells are grown in the presence of thiamine and 5-(2-hydroxyethyl)-4-methylthiazole. The thi3 gene involved in the biosynthesis of the pyrimidine moiety, is also regulated by thiamine [Maundrell, J. Biol. Chem. 265 (1990) 10857-10864; Schweingruber et al., Curr. Genet. 19 (1991) 249-254]. We previously identified and analyzed four regulatory genes (tnr1, tnr2, tnr3, and thi1) that are responsible for the regulation of thi3 [Schweingruber et al., Genetics (1992) in press]. Mutants defective in these regulatory genes affect expression of thi2 in a similar way to thi3. This indicates that biosynthesis of the pyrimidine and thiazole moieties are under common genetic control in S. pombe.     

Conjugation-induced lysis of Schizosaccharomyces pombe.

About 15% of the conjugating cells of Schizosaccharomyces pombe were observed to lyse spontaneously during the conjugation process. Lysis occurred at the site of union.     

Tpr1, a Schizosaccharomyces pombe protein involved in potassium transport.

The Schizosaccharomyces pombe Tpr1 was isolated as suppressor of the Saccharomyces cerevisiae Delta trk1,2 potassium uptake deficient phenotype. Tpr1, for tetratrico peptide repeat, encodes a 1039 amino acid residues protein with several reiterated TPR units displaying significant homology to p150(TSP), a recently identified phosphoprotein of mouse, to S. cerevisiae CTR9 and to related sequences of human, Caenorhabditis elegans, Methanoccocus jannaschii and Arabidopsis thaliana. Expression of Tpr1 restored growth on 0.2 mM K(+) media, induced K(+) transport with a K(T) of 4.6 mM and resumed inward currents of -90 pA at -250 mV (pH 7.2) conducting K(+) and other alkali-metal ions. The tetratrico peptide repeat is a degenerate motif of 34 amino acids that is repeated several times within TPR-containing proteins and has been suggested to mediate protein-protein interactions. The sequence and putative binding properties of Tpr1 suggest the protein unlikely as transporter but involved in the enhancement of K(+) uptake via conventional carriers.     

Cytochrome c from Schizosaccharomyces pombe. 2. Amino-acid sequence.

The amino acid sequence of Schizosaccharomyces pombe cytochrome c has been established by automatic degradation of the protein and by manual degradation of fragments obtained by cyanogen bromide cleavage and chymotryptic digestion. The chymotryptic peptides were aligned by homology with other known cytochrome c sequences. The protein is 108 residues long, with a four-residue amino-terminal tail. It has only one methionine residue and differs from other fungal cytochromes c in lacking the one-residue deletion at the C-terminal end. After a cyanogen bromide step, an unexpected cleavage of the peptide chain before a cysteine residue was observed. This is ascribed to formation of a dehydroalanyl residue during an incomplete S-carboxymethylation of the apoprotein, and subsequent cleavage under acidic conditions. Experimental evidence is presented in favour of the proposed mechanisms.     

Effect of 2-deoxyglucose on Schizosaccharomyces pombe

Megnet, Roland (Institut für Allgemeine Mikrobiologie der Universit?t, Bern, Switzerland). Effect of 2-deoxyglucose on Schizosaccharomyces pombe. J. Bacteriol. 90:1032-1035. 1965.-Cultivation of Schizosaccaromyces pombe in a medium containing 2-deoxyglucose (100 mug/ml) results in the death of the cells after an initial period of apparently normal growth. At higher deoxyglucose concentration (400 mug/ml), the cells die immediately after inoculation. Only growing cells are killed, and microscopic inspection of the cultures reveals cell-wall fragments of lysed cells. A mutant resistant to 2-deoxyglucose, which cannot use glucose as a carbon source, was found to be partially deficient in hexokinase. The data constitute evidence for the inhibition of some reaction(s) in the synthesis of cell-wall polysaccharides by metabolites of 2-deoxyglucose in this organism.