Yuko Giga-Hama

Foreign Gene Expression in Fission Yeast: Schizosaccharomyces pombe

1. Fission Yeast Schizosaccharomycespombe: An Attractive Host for Heterologous Protein Production.- 2. Constitution of Expression Vectors.- 3. Exploiting the Thiamine Biosynthetic Gene nmtl for Regulated Expression of Heterologous DNAs in Fission Yeast.- 4. Development of Constitutive Expression Vector for the Highest Production Level of Heterologous Proteins in Fission Yeast Schizosaccharomycespombe.- 5. Overexpression of Proteins with Seven Transmembrane Spanning Segments.- 6. Targeting and Isolation of Tagged Membrane Proteins.- 7. Expression of a Human Cytochrome P450 Form in Schizosaccharomyces pombe: Comparison with Expression in Saccharomyces cerevisiae.- 8. Human Papillomavirus Capsid Protein-pREP in Schizosaccharomyces pombe: Efficient Assembly of the Viral Capsid Protein in S. pombe and S. cerevisiae.- 9. Expression, Purification, and Analyses of Cell-Cycle Regulatory Proteins in S. pombe.- 10. Expression and Secretion of Mammalian Proteins in Schizosaccharomyces pombe.- 11. Secretion of Human Interleukin-6 Using the P-Factor Secretion Signal in Schizosaccharomyces pombe.- Color Figures.

Expression system for foreign genes using the fission yeast Schizosaccharomyces pombe.

Foreign‐gene expression systems using mammalian cells, Escherichia coli, insect cells, yeast and other organisms as hosts have been developed. The demand for protein‐production systems will be further increased in basic research, medical science and the biotechnological industry. Systems using the fission yeast Schizosaccharomyces pombe as a host have only recently received attention. The advantages of this yeast, which is more advanced evolutionarily than other types of yeast, the expression vectors available and examples of heterologous protein produced with this system, are reviewed here.

Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Δ, vps22Δ, and vps34Δ, were determined on account of their hGH secretion efficiency. The mutant vps10Δ was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.

Zinc Finger Protein Prz1 Regulates Ca2+ but Not Cl− Homeostasis in Fission Yeast IDENTIFICATION OF DISTINCT BRANCHES OF CALCINEURIN SIGNALING PATHWAY IN FISSION YEAST

Calcineurin is an important mediator that connects the Ca2+-dependent signaling to various cellular responses in a wide variety of cell types and organisms. In budding yeast, activated calcineurin exerts its function mainly by regulating the Crz1p/Tcn1 transcription factor. Here, we cloned the fission yeast prz1 + gene, which encodes a zinc finger transcription factor highly homologous to Crz1/Tcn1. Similar to the results in budding yeast, calcineurin dephosphorylated Prz1 and resulted in the trans-location of Prz1 from the cytoplasm to the nucleus. Prz1 expression was stimulated by high extracellular Ca2+ in a calcineurin-dependent fashion. However, unlike in budding yeast, the prz1-null cells did not show any phenotype similar to those previously reported in calcineurin deletion such as aberrant cell morphology, mating defect, or hypersensitivity to Cl−. Instead, theprz1-null cells showed hypersensitivity to Ca2+, consistent with a dramatic decrease in transcription of Pmc1 Ca2+ pump. Interestingly, overexpression of Prz1 did not suppress the Cl− hypersensitivity of calcineurin deletion, and overexpression of Pmp1 MAPK phosphatase suppressed the Cl− hypersensitivity of calcineurin deletion but not the Ca2+ hypersensitivity of prz1 deletion. In addition, mutations in theits2 + /cps1 + ,its8 + , andits10 + /cdc7 + genes that showed synthetic lethal genetic interaction with calcineurin deletion did not exhibit synthetic lethality with the prz1 deletion. Our results suggest that calcineurin activates at least two distinct signaling branches, i.e. the Prz1-dependent transcriptional regulation and an unknown mechanism, which functions antagonistically with the Pmk1 MAPK pathway.

Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe.

Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of erg31(+) or erg32(+) did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in ergDelta mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6Delta but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the ergDelta mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins.

Schizosaccharomyces pombe minimum genome factory.

Various systems for the production of useful proteins have been developed using the fission yeast Schizosaccharomyces pombe as a host, and some are now being used commercially. It is necessary, however, to improve the system further for the production of low‐cost chemicals and commodities, so that the host becomes more economical and productive and can be widely used for the production of different molecules. We hypothesized that many S. pombe genes are not necessary under nutrient‐rich growth conditions; or rather, they serve only to waste energy when seen from the viewpoint of protein production, because their products are necessary only for adaptation to different environments. Thus we have tried to create S. pombe mutants that are dedicated to heterologous protein production by deleting as many non‐essential genes as possible. Putative essential genes were mapped using the genome information of S. pombe. The transcriptome of gene disruptants was analysed using microarrays and, using this system, a new promoter was identified. The method (called the Latour method) has been developed to delete efficiently a large region from the chromosome, resulting in the establishment of mutant strains lacking approx. 500 kb of genetic material. New experimental strains auxotrophic for six nutrients were established that were conveniently used for co‐expression of proteins using multiple plasmids. An efficient transformation method has also been developed that is useful for investigating heterologous protein production in a variety of strains. Incidentally, in heterologous protein production systems, products are often degraded, leading to a decline in production efficiency. Thus, to examine heterologous protein production, we created 52 S. pombe mutant strains in each of which a single protease gene was destroyed. We also successfully constructed strains in which multiple protease genes were disrupted. As a result, it was shown that the production of a model protein, human growth hormone, was increased in this strain. Furthermore, we obtained many strains that lacked genes related to glucose metabolism, intracellular transport or biosynthesis of sugar chains. The present minireview covers the results of functional analysis of these strains. By preparing strains in which large chromosomal regions have been deleted and then combining strains defective in various functional genes, the establishment of effective hosts will become possible.

Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system

The fission yeast Schizosaccharomyces pombe is a particularly useful model for studying the function and regulation of genes from higher eukaryotes. The genome of Sc. pombe has been sequenced, and DNA microarray, proteome and transcriptome analyses have been carried out. Among the well‐characterized yeast species, Sc. pombe is considered an attractive host for the production of heterologous proteins. Expression vectors for high‐level expression in Sc. pombe have been developed and many foreign proteins have been successfully expressed. However, further improvements in the protein‐expressing host systems are still required for the production of heterologous proteins involved in post‐translational modification, metabolism and intracellular trafficking. This minireview focuses on recent advances in heterologous protein production by use of engineered fission‐yeast strains.

Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe.

The creation of protease-deficient mutants to avoid product degradation is one of the current strategies employed to improve productivity and secretion efficiency of heterologous protein expression. We previously constructed a set of single protease-deficient mutants of the fission yeast Schizosaccharomyces pombe by respective disruption of 52 protease genes, and we succeeded in confirming useful disruptants (Idiris et al., Yeast 23:83–99, 2006). In the present study, we attempted multiple deletions of 13 protease genes, single deletions of which were previously confirmed as being beneficial for reducing extracellular product degradation. Using PCR-based gene replacement, a series of multiple deletion strains was constructed by multiple disruption of a maximum of seven protease genes. Effects of the resultant multiple deletion strains on heterologous expression were then measured by practical expression of a proteolytically sensitive model protein, the human growth hormone (hGH). Time profiles of hGH secretion from each resultant mutant demonstrated significantly enhanced hGH productivity with processing of the multiple protease deletions. The data clearly indicated that disruption of multiple protease genes in the fission yeast is an effective method for controlling proteolytic degradation of heterologous proteins particularly susceptible to proteases.

Characterization of end4+, a gene required for endocytosis in Schizosaccharomyces pombe.

To understand endocytic trafficking in Schizosaccharomyces pombe, we constructed an end4 disruption mutant. The end4+ gene encodes a protein homologous to Sla2p/End4p, which is essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We characterized the fission yeast mutant end4Δ as well as ypt7Δ, which is deficient in vacuolar fusion and, hence, endocytosis. The delivery of FM4‐64 to the vacuolar membrane, accumulation of Lucifer yellow CH and internalization of plasma membrane protein Map3–GFP were inhibited in the end4 mutant. Deletion of end4 resulted in pleiotropic phenotypes consistent with F‐actin depolarization, including high temperature sensitivity, abnormal morphology and mating defects. Extensive missorting of carboxypeptidase Y was detected in the ypt7 mutant; however, little missorting was detected in the end4 mutant. These results indicate that End4p is essential for the internalization process and Ypt7p affects endocytosis at a post‐internalization step after the intersection of the endocytic and the vacuolar protein‐sorting pathways in fission yeast. Copyright

A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast

The technologies for chromosome modification developed to date are not satisfactorily universal, owing to the typical requirements for special enzymes and sequences. In the present report, we propose a new approach for chromosome modification in Schizosaccharomyces pombe that does not involve any special enzymes or sequences. This method, designated the ‘Latour system’, has wide applicability with extremely high efficiency, although both the basic principle and the operation are very simple. We demonstrate the ability of the Latour system to discriminate essential genes, with a long chromosomal area of 100 kb containing 33 genes deleted simultaneously and efficiently. Since no foreign sequences are retained after deletion using the Latour system, this system can be repeatedly applied at other sites. Provided that a negative selectable marker is available, the Latour system relies solely upon homologous recombination, which is highly conserved in living organisms. For this reason, it is expected that the system will be applicable to various yeasts.