A novel meiosis-specific protein of fission yeast, Meu13p, promotes homologous pairing independently of homologous recombination.

Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation. We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination. Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe. Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not. We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion. Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe. Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.     

Molecular and Genetic Analysis of Rec103, an Early Meiotic Recombination Gene in Yeast

In the yeast Saccharomyces cerevisiae at least 10 genes are required to begin meiotic recombination. A new early recombination gene REC103 is described in this paper. It was initially defined by the rec103-1 mutation found in a selection for mutations overcoming the spore inviability of a rad52 spo13 haploid strain. Mutations in REC103 also rescue rad52 in spo13 diploids. rec103 spo13 strains produce viable spores; these spores show no evidence of meiotic recombination. rec103 SPO13 diploids produce no viable spores, consistent with the loss of recombination. Mutations in REC103 do not affect mitotic recombination, growth, or repair. These phenotypes are identical to those conferred by mutations in several other early meiotic recombination genes (e.g., REC102, REC104, REC114, MEI4, MER2, and SPO11). REC103 maps to chromosome VII between ADE5 and RAD54. Cloning and sequencing of REC103 reveals that REC103 is identical to SKI8, a gene that depresses the expression of yeast double-stranded (``killer') (ds)RNA viruses. REC103/SKI8 is transcribed in mitotic cells and is induced ~15-fold in meiosis. REC103 has 26% amino acid identity to the Schizosaccharomyces pombe rec14(+) gene; mutations in both genes confer similar meiotic phenotypes, suggesting that they may play similar roles in meiotic recombination.     

Linear element-independent meiotic recombination in Schizosaccharomyces pombe

Most organisms form protein-rich, linear, ladder-like structures associated with chromosomes during early meiosis, the synaptonemal complex. In Schizosaccharomyces pombe, linear elements (LinEs) are thread-like, proteinacious chromosome-associated structures that form during early meiosis. LinEs are related to axial elements, the synaptonemal complex precursors of other organisms. Previous studies have led to the suggestion that axial structures are essential to mediate meiotic recombination. Rec10 protein is a major component of S. pombe LinEs and is required for their development. In this report we study recombination in a number of rec10 mutants, one of which (rec10-155) does not form LinEs, but is predicted to encode a truncated Rec10 protein. This mutant has levels of crossing over and gene conversion substantially higher than a rec10 null mutant (rec10-175) and forms cytologically detectable Rad51 foci indicative of meiotic recombination intermediates. These data demonstrate that while Rec10 is required for meiotic recombination, substantial meiotic recombination can occur in rec10 mutants that do not form LinEs, indicating that LinEs per se are not essential for all meiotic recombination.     

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.     

Meiotically Induced Rec7 and Rec8 Genes of Schizosaccharomyces Pombe

The Schizosaccharomyces pombe rec7 and rec8 genes, which are required for meiotic intragenic recombination but not for mitotic recombination, have been cloned and their DNA sequences determined. Genetic and physical analyses demonstrated that the cloned fragments contained the rec genes rather than rec mutation suppressors. A 1.6-kb DNA fragment contained a functional rec7 gene, and a 2.1-kb fragment contained a functional rec8 gene. The nucleotide sequences of these fragments revealed open reading frames predicting 249 amino acids for the rec7 gene product and 393 amino acids for the rec8 gene product. Northern hybridization analysis showed that both rec gene mRNAs were detectable only at 2-3 hr after induction of meiosis. The absence of these mRNAs in mitosis and their disappearance at 4 hr and later in meiosis suggest that the rec7 and rec8 gene products may be involved primarily in the early steps of meiotic recombination in S. pombe.     

Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.     

Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue.

Using the cloned Saccharomyces cerevisiae YPT1 gene as hybridization probe, a gene, designated ypt2, was isolated from the fission yeast Schizosaccharomyces pombe and found to encode a 200 amino acid long protein most closely related to the ypt branch of the ras superfamily. Disruption of the ypt2 gene is lethal. The bacterially produced ypt2 gene product is shown to bind GTP. A region of the ypt2 protein corresponding to but different from the 'effector region' of ras proteins is also different from that of ypt1 proteins of different species but identical to the 'effector loop' of the S.cerevisiae SEC4 gene product, a protein known to be required for vesicular protein transport. The S.pombe ypt2 gene under control of the S.cerevisiae GAL10 promoter is able to suppress the temperature-sensitive phenotype of a S. cerevisiae sec4 mutant, indicating a functional similarity of these GTP-binding proteins from the two very distantly related yeasts.     

Nonrandom homolog segregation at meiosis I in Schizosaccharomyces pombe mutants lacking recombination

Physical connection between homologous chromosomes is normally required for their proper segregation to opposite poles at the first meiotic division (MI). This connection is generally provided by the combination of reciprocal recombination and sister-chromatid cohesion. In the absence of meiotic recombination, homologs are predicted to segregate randomly at MI. Here we demonstrate that in rec12 mutants of the fission yeast Schizosaccharomyces pombe, which are devoid of meiosis-induced recombination, homologs segregate to opposite poles at MI 63% of the time. Residual, Rec12-independent recombination appears insufficient to account for the observed nonrandom homolog segregation. Dyad asci are frequently produced by rec12 mutants. More than half of these dyad asci contain two viable homozygous-diploid spores, the products of a single reductional division. This set of phenotypes is shared by other S. pombe mutants that lack meiotic recombination, suggesting that nonrandom MI segregation and dyad formation are a general feature of meiosis in the absence of recombination and are not peculiar to rec12 mutants. Rec8, a meiosis-specific sister-chromatid cohesin, is required for the segregation phenotypes displayed by rec12 mutants. We propose that S. pombe possesses a system independent of recombination that promotes homolog segregation and discuss possible mechanisms.     

Rec25 and Rec27, novel linear-element components, link cohesin to meiotic DNA breakage and recombination

Meiosis is a specialized nuclear division by which sexually reproducing diploid organisms generate haploid gametes. Recombination between homologous chromosomes facilitates accurate meiotic chromosome segregation and is initiated by DNA double-strand breaks (DSBs) made by the conserved topoisomerase-like protein Spo11 (Rec12 in fission yeast), but DSBs are not evenly distributed across the genome. In Schizosaccharomyces pombe, proteinaceous structures known as linear elements (LinEs) are formed during meiotic prophase. The meiosis-specific cohesin subunits Rec8 and Rec11 are essential for DSB formation in some regions of the genome, as well as for formation of LinEs or the related synaptonemal complex (SC) in other eukaryotes. Proteins required for DSB formation decorate LinEs, and mutants lacking Rec10, a major component of LinEs, are completely defective for recombination. Although recombination may occur in the context of LinEs, it is not well understood how Rec10 is loaded onto chromosomes. We describe two novel components of LinEs in fission yeast, Rec25 and Rec27. Comparisons of rec25Delta, rec27Delta, and rec10Delta mutants suggest multiple pathways to load Rec10. In the major pathway, Rec10 is loaded, together with Rec25 and Rec27, in a Rec8-dependent manner with subsequent region-specific effects on recombination.     

A functional homologue of the RNA1 gene product in Schizosaccharomyces pombe: purification, biochemical characterization, and identification of a leucine-rich repeat motif.

The RNA1 gene from Saccharomyces cerevisiae is defined by the temperature-sensitive rna1-1 mutation that interferes with the maturation and/or nucleocytoplasmic transport of RNA. We describe the purification of a 44-kDa protein from the evolutionary distant fission yeast Schizosaccharomyces pombe and the cloning and sequence analysis of the corresponding gene. Although this protein shares only 42% sequence identity with the RNA1 gene product, it represents a functional homologue because the expression of the S. pombe gene in S. cerevisiae complements the rna1-1 defect. Disruption in S. pombe of the gene encoding the 44-kDa protein, for which we propose the name S. pombe rna1p, reveals that it is essential for growth. Our analysis of purified S. pombe rna1p represents the first biochemical characterization of an RNA1 gene product and reveals that it is a monomeric protein of globular shape. Cell fractionation and immunofluorescence microscopy indicate that rna1p is a cytoplasmic protein possibly enriched in the nuclear periphery. We identify a sequence motif of 29 residues, which is rich in leucine and repeated eight times both in S. pombe and in S. cerevisiae rna1p. Similar leucine-rich repeats present in a series of other proteins, e.g., the mammalian ribonuclease/angiogenin inhibitor, adenylyl cyclase from S. cerevisiae, the toll protein from Drosophila melanogaster, and the sds22 protein phosphatase regulatory subunit from S. pombe, are thought to be involved in protein-protein interactions. Thus rna1p may act as a scaffold protein possibly interacting in the nuclear periphery with a protein ligand that could be associated with exported RNA.     

Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation.

Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme. Inhibition of sterol synthesis in S. cerevisiae and S. pombe cells or in cultured human fibroblasts by treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor lovastatin resulted in elevated levels of squalene synthetase mRNA in all three cell types.     

A recombination repair gene of Schizosaccharomyces pombe, rhp57, is a functional homolog of the Saccharomyces cerevisiae RAD57 gene and is phylogenetically related to the human XRCC3 gene

To identify Schizosaccharomyces pombe genes involved in recombination repair, we identified seven mutants that were hypersensitive to both methyl methanesulfonate (MMS) and gamma-rays and that contained mutations that caused synthetic lethality when combined with a rad2 mutation. One of the mutants was used to clone the corresponding gene from a genomic library by complementation of the MMS-sensitive phenotype. The gene obtained encodes a protein of 354 amino acids whose sequence is 32% identical to that of the Rad57 protein of Saccharomyces cerevisiae. An rhp57 (RAD57 homolog of S. pombe) deletion strain was more sensitive to MMS, UV, and gamma-rays than the wild-type strain and showed a reduction in the frequency of mitotic homologous recombination. The MMS sensitivity was more severe at lower temperature and was suppressed by the presence of a multicopy plasmid bearing the rhp51 gene. An rhp51 rhp57 double mutant was as sensitive to UV and gamma-rays as an rhp51 single mutant, indicating that rhp51 function is epistatic to that of rhp57. These characteristics of the rhp57 mutants are very similar to those of S. cerevisiae rad57 mutants. Phylogenetic analysis suggests that Rhp57 and Rad57 are evolutionarily closest to human Xrcc3 of the RecA/Rad51 family of proteins.     

Functional analysis of chimerical plasma membrane H+-ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe

The plasma membrane H⁺-ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H⁺-ATPase-depleted cells of the budding yeast Saccharomyces cerevisiae , even after deletion of the enzyme's carboxy terminus. Functional chimerical H⁺-ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site-directed mutagenesis of the H⁺-ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H⁺-ATPase. The reverse Ala-598 →Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H⁺-ATPase substituted with alanine were enlarged and grew more slowly than wild-type cells. ATPase activity showed a more alkaline pH optimum, lower K _m values for MgATP and decreased V _max compared with wild-type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose-starved cells, indicating that the S. pombe H⁺-ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.