Vesicle-mediated protein transport pathways to the vacuole in Schizosaccharomyces pombe

The vacuole of Saccharomyces cerevisiae plays essential roles not only for osmoregulation and ion homeostasis but also down-regulation (degradation) of cell surface proteins and protein and organellar turnover. Genetic selections and genome-wide screens in S. cerevisiae have resulted in the identification of a large number of genes required for delivery of proteins to the vacuole. Although the complete genome sequence of the fission yeast Schizosaccharomyces pombe has been reported, there have been few reports on the proteins required for vacuolar protein transport and vacuolar biogenesis in S. pombe. Recent progress in the S. pombe genome project of has revealed that most of the genes required for vacuolar biogenesis and protein transport are conserved between S. pombe and S. cerevisiae. This suggests that the basic machinery of vesicle-mediated protein delivery to the vacuole is conserved between the two yeasts. Identification and characterization of the fission yeast counterparts of the budding yeast Vps and Vps-related proteins have facilitated our understanding of protein transport pathways to the vacuole in S. pombe. This review focuses on the recent advances in vesicle-mediated protein transport to the vacuole in S. pombe.     

The primary structure of the alcohol dehydrogenase gene from the fission yeast Schizosaccharomyces pombe

We have cloned and sequenced the alcohol dehydrogenase gene of the fission yeast Schizosaccharomyces pombe. The gene was isolated by transformation and complementation of a Saccharomyces cerevisiae strain which lacked functional alcohol dehydrogenase with an S. pombe gene bank constructed in the autonomously replicating yeast plasmid YEp13. Southern hybridization analysis indicates that S. pombe contains only one alcohol dehydrogenase gene. The structural region of the gene is 50% homologous to the alcohol dehydrogenase encoding genes of the budding yeast S. cerevisiae. The gene exhibits a very strong codon usage bias; with the set of predominantly used codons generally resembling that which S. cerevisiae employs preferentially. All of the differences in codon usage bias between S. pombe and S. cerevisiae are in the direction of greater G + C content in S. pombe codons. It is argued that this observation supports the hypothesis that selection toward uniform codon-anticodon binding energies contributes to codon usage bias and that the optimum binding energy is, on the average, higher in S. pombe than S. cerevisiae.     

Homologous mRNA 3'' end formation in fission and budding yeast.

Sequences resembling polyadenylation signals of higher eukaryotes are present downstream of the Schizosaccharomyces pombe ura4+ and cdc10+ coding regions and function in HeLa cells. However, these and other mammalian polyadenylation signals are inactive in S. pombe. Instead, we find that polyadenylation signals of the CYC1 gene of budding yeast Saccharomyces cerevisiae function accurately and efficiently in fission yeast. Furthermore, a 38 bp deletion which renders this RNA processing signal non-functional in S. cerevisiae has the equivalent effect in S. pombe. We demonstrate that synthetic pre-mRNAs encoding polyadenylation sites of S. pombe genes are accurately cleaved and polyadenylated in whole cell extracts of S. cerevisiae. Finally, as is the case in S. cerevisiae, DNA sequences encoding regions proximal to the S. pombe mRNA 3' ends are found to be extremely AT rich; however, no general sequence motif can be found. We conclude that although fission yeast has many genetic features in common with higher eukaryotes, mRNA 3' end formation is significantly different and appears to be formed by an RNA processing mechanism homologous to that of budding yeast. Since fission and budding yeast are evolutionarily divergent, this lower eukaryotic mechanism of mRNA 3' end formation may be generally conserved.     

The Rpb4 Subunit of Fission Yeast Schizosaccharomyces pombe RNA Polymerase II Is Essential for Cell Viability and Similar in Structure to the Corresponding Subunits of Higher Eukaryotes

Both the gene and the cDNA encoding the Rpb4 subunit of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe. The cDNA sequence indicates that Rpb4 consists of 135 amino acid residues with a molecular weight of 15,362. As in the case of the corresponding subunits from higher eukaryotes such as humans and the plant Arabidopsis thaliana, Rpb4 is smaller than RPB4 from the budding yeast Saccharomyces cerevisiae and lacks several segments, which are present in the S. cerevisiae RPB4 subunit, including the highly charged sequence in the central portion. The RPB4 subunit of S. cerevisiae is not essential for normal cell growth but is required for cell viability under stress conditions. In contrast, S. pombe Rpb4 was found to be essential even under normal growth conditions. The fraction of RNA polymerase II containing RPB4 in exponentially growing cells of S. cerevisiae is about 20%, but S. pombe RNA polymerase II contains the stoichiometric amount of Rpb4 even at the exponential growth phase. In contrast to the RPB4 homologues from higher eukaryotes, however, S. pombe Rpb4 formed stable hybrid heterodimers with S. cerevisiae RPB7, suggesting that S. pombe Rpb4 is similar, in its structure and essential role in cell viability, to the corresponding subunits from higher eukaryotes. However, S. pombe Rpb4 is closer in certain molecular functions to S. cerevisiae RPB4 than the eukaryotic RPB4 homologues.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

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.     

Recombinational repair in Schizosaccharomyces pombe: role in maintaining genomic integrity

Recombinational repair was first detected in budding yeast Saccharomyces cerevisiae and was also studied in fission yeast Schizosaccharomyces pombe over the recent decade. The discovery of Sch. pombe homologs of the S. cerevisiae RAD52 genes made it possible not only to identify and to clone their vertebrate counterparts, but also to study in detail the role of DNA recombination in certain cell processes. For instance, recombinational repair was shown to play a greater role in maintaining genome integrity in fission yeast and in vertebrates compared with S. cerevisiae. The present state of the problem of recombinational double-strand break repair in fission yeast is considered with a focus on comparisons between Sch. pombe and higher eukaryotes. The role of double-strand break repair in maintaining genome stability is discussed.     

Conservation of the prion properties of Ure2p through evolution

The yeast inheritable [URE3] element corresponds to a prion form of the nitrogen catabolism regulator Ure2p. We have isolated several orthologous URE2 genes in different yeast species: Saccharomyces paradoxus, S. uvarum, Kluyveromyces lactis, Candida albicans, and Schizosaccharomyces pombe. We show here by in silico analysis that the GST-like functional domain and the prion domain of the Ure2 proteins have diverged separately, the functional domain being more conserved through the evolution. The more extreme situation is found in the two S. pombe genes, in which the prion domain is absent. The functional analysis demonstrates that all the homologous genes except for the two S. pombe genes are able to complement the URE2 gene deletion in a S. cerevisiae strain. We show that in the two most closely related yeast species to S. cerevisiae, i.e., S. paradoxus and S. uvarum, the prion domains of the proteins have retained the capability to induce [URE3] in a S. cerevisiae strain. However, only the S. uvarum full-length Ure2p is able to behave as a prion. We also show that the prion inactivation mechanisms can be cross-transmitted between the S. cerevisiae and S. uvarum prions.     

Versatile use of Schizosaccharomyces pombe plasmids in Saccharomyces cerevisiae

The two model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe appear to have diverged 1000 million years ago. Here, we describe that S.?pombe vectors can be propagated efficiently in S.?cerevisiae as pUR19 derivatives, and the pREP and pJR vector series carrying the S.?cerevisiae LEU2 or the S.?pombe ura4(+) selection marker are maintained in S.?cerevisiae cells. In addition, genes transcribed from the S.?pombe nmt1(+) promoter and derivatives are expressed in budding yeast. Thus, S.?pombe vectors can be used as shuttle vectors in S.?cerevisiae and S.?pombe. Our finding greatly facilitates the testing for functional orthologs of protein families and simplifies the cloning of new S.?pombe plasmids by using the highly efficient in vivo homologous recombination activity of S.?cerevisiae.     

Chronological aging-induced apoptosis in yeast

Saccharomyces cerevisiae is the simplest among the major eukaryotic model organisms for aging and diseases. Longevity in the chronological life span paradigm is measured as the mean and maximum survival period of populations of non-dividing yeast. This paradigm has been used successfully to identify several life-regulatory genes and three evolutionary conserved pro-aging pathways. More recently, Schizosaccharomyces pombe has been shown to age chronologically in a manner that resembles that of S. cerevisiae and that depends on the activity of the homologues of two pro-aging proteins previously identified in the budding yeast. Both yeast show features of apoptotic death during chronological aging. Here, we review some fundamental aspects of the genetics of chronological aging and the overlap between yeast aging and apoptotic processes with particular emphasis on the identification of an aging/death program that favors the dedifferentiation and regrowth of a few better adapted mutants generated within populations of aging S. cerevisiae. We also describe the use of a genome-wide screening technique to gain further insights into the mechanisms of programmed death in populations of chronologically aging S. cerevisiae.     

Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids.     

裂殖酵母作为外源基因表达系统

虽然裂殖酵母与酿酒酵母同属于子囊真菌,但比其它的酵母相比,裂殖酵母与更高等的真核细胞有许多相似的性质,使得裂殖酵母在分子生物学研究中成为一种提供信息的、准确的真核实验模型.它在外源基因表达方面同样具有前景.主要介绍了裂殖酵母的优点,其表达载体的性质,以及外源蛋白表达的例子.     

Stress-specific role of fission yeast Gcn5 histone acetyltransferase in programming a subset of stress response genes

Gcn5 is a coactivator protein that contributes to gene activation by acetylating specific lysine residues within the N termini of histone proteins. Gcn5 has been intensively studied in the budding yeast, Saccharomyces cerevisiae, but the features of genes that determine whether they require Gcn5 during activation have not been conclusively clarified. To allow comparison with S. cerevisiae, we have studied the genome-wide role of Gcn5 in the distantly related fission yeast, Schizosaccharomyces pombe. We show that Gcn5 is specifically required for adaptation to KCl- and CaCl(2)-mediated stress in S. pombe. We have characterized the genome-wide gene expression responses to KCl stress and show that Gcn5 is involved in the regulation of a subset of stress response genes. Gcn5 is most clearly associated with KCl-induced genes, but there is no correlation between Gcn5 dependence and the extent of their induction. Instead, Gcn5-dependent KCl-induced genes are specifically enriched in four different DNA motifs. The Gcn5-dependent KCl-induced genes are also associated with biological process gene ontology terms such as carbohydrate metabolism, glycolysis, and nicotinamide metabolism that together constitute a subset of the ontology parameters associated with KCl-induced genes.     

A new recombinational DNA repair gene from Schizosaccharomyces pombe with homology to Escherichia coli RecA.

A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55(+) (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an M(r) of 38,000. The rhp55Delta mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Delta cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55(+) acts in one DNA repair pathway with rhp51(+) and rhp54(+), homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55(+) is in a different epistasis group for repair of UV-, MMS-, or gamma-ray-induced DNA damage than is rad22(+), a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55(+) is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved.     

Cloning and sequence determination of the gene encoding the largest subunit of the fission yeast Schizosaccharomyces pombe RNA polymerase I

The gene encoding the largest subunit of RNA polymerase I (SPRPA190) was cloned from the fission yeast Schizosaccharomyces pombe by cross-hybridization with a probe containing part of the corresponding Saccharomyces cerevisiae gene RPA190. The SPRPA190 gene is present in a single copy per haploid genome and is essential for cell growth. The polypeptide encoded by this gene, as deduced from the nucleotide sequence of the uninterrupted coding frame, consists of 1689 amino acids and its calculated Mr is 189,300. The amino acid identity between the subunits of the two yeast species is 50%. Amino acid sequence conservation covers the regions previously suggested to be functionally important for the S. cerevisiae enzyme. In addition, two markedly hydrophilic regions recognized in the S. cerevisiae polypeptide can also be recognized in the S. pombe polypeptide in approximately the same positions, even though the amino acid sequences in these regions are diverged from each other. In the 5'-flanking region of the gene, several nucleotide sequence elements are detected which are also found in the two S. pombe ribosomal protein genes so far sequenced.