Endocytosis is essential for dynamic translocation of a syntaxin 1 orthologue during fission yeast meiosis.

Syntaxin is a component of the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, which is responsible for fusion of membrane vesicles at the target membrane. The fission yeast syntaxin 1 orthologue Psy1 is essential for both vegetative growth and spore formation. During meiosis, Psy1 disappears from the plasma membrane (PM) and dramatically relocalizes on the nascent forespore membrane, which becomes the PM of the spore. Here we report the molecular details and biological significance of Psy1 relocalization. We find that, immediately after meiosis I, Psy1 is selectively internalized by endocytosis. In addition, a meiosis-specific signal induced by the transcription factor Mei4 seems to trigger this internalization. The internalization of many PM proteins is facilitated coincident with the initiation of meiosis, whereas Pma1, a P-type ATPase, persists on the PM even during the progression of meiosis II. Ergosterol on the PM is also important for the internalization of PM proteins in general during meiosis. We consider that during meiosis in Schizosaccharomyces pombe cells, the characteristics of endocytosis change, thereby facilitating internalization of Psy1 and accomplishing sporulation.

New insights into galactose metabolism by Schizosaccharomyces pombe: isolation and characterization of a galactose-assimilating mutant.

The fission yeast Schizosaccharomyces pombe cannot use galactose as a carbon or energy source, and little is known about galactose metabolism in this species. Here we report isolation of a galactose-assimilating mutant that grows on a medium containing galactose as a sole carbon source through use of a proofreading-deficient DNA polymerase δ variant encoded by cdc6-1. Based on comparative analysis of gene expression profiles in the wild-type and the mutant (FG2-8), we found that SPBPB2B2.10c (gal7+), SPBPB2B2.12c (gal10+) and SPBPB2B2.13 (gal1+), homologous to Saccharomyces cerevisiae GAL7, GAL10 and GAL1, respectively, and SPBPB2B2.08, SPBPB2B2.09c, and SPBPB2B2.11 that localize close to the gal genes, were highly expressed and dramatically induced by addition of galactose. The gal7Δ strain, carrying an integrated ura4+ marker at the gal7+ locus, grew on 5-fluoroorotic acid (5-FOA)-containing medium. In contrast, the FG2-8 gal7Δ strain could not grow on 5-FOA medium. In addition, expression of gal7+, SPBPB2B2.13, gal10+ and gal1+ genes increased in the wild-type strain when carried on a vector, and these transformants grew on galactose medium. We suggest that gal7+, gal10+, and gal1+ are localized close to a chromosomal terminal repressed by gene silencing in S. pombe. In contrast, gene silencing was defective in the FG2-8 strain making galactose assimilation possible.

Schizosaccharomyces pombe calmodulin, Cam1, plays a crucial role in sporulation by recruiting and stabilizing the spindle pole body components responsible for assembly of the forespore membrane.

Calmodulin in Schizosaccharomyces pombe is encoded by the cam1(+) gene, which is indispensable for both vegetative growth and sporulation. Here, we report how Cam1 functions in spore formation. We found that Cam1 preferentially localized to the spindle pole body (SPB) during meiosis and sporulation. Formation of the forespore membrane, a precursor of the plasma membrane in spores, was blocked in a missense cam1 mutant, which was viable but unable to sporulate. Three SPB proteins necessary for the onset of forespore membrane formation, Spo2, Spo13, and Spo15, were unable to localize to the SPB in the cam1 mutant although five core SPB components that were tested were present. Recruitment of Spo2 and Spo13 is known to require the presence of Spo15 in the SPB. Notably, Spo15 was unstable in the cam1 mutant, and as a result, SPB localization of Spo2 and Spo13 was lost. Overexpression of Spo15 partially alleviated the sporulation defect in the cam1 mutant. These results indicate that calmodulin plays an essential role in forespore membrane formation by stably maintaining Spo15, and thus Spo2 and Spo13, at the SPB in meiotic cells.

Development of valuable yeast strains using a novel mutagenesis technique for the effective production of therapeutic glycoproteins.

Yeast cells producing mammalian-type N-linked oligosaccharide show severe growth defects and the decreased protein productivity because of the disruption of yeast-specific glycosyltransferases. This decreased protein productivity in engineered yeast strains is an obstacle to the development of efficient glycoprotein production in yeast. For economic and effective synthesis of such therapeutic glycoproteins in yeast, the development of appropriate strains is highly desirable. We applied a novel mutagenesis technique that utilized the proofreading-deficient DNA polymerase delta variant encoded by the pol3-01 gene of Saccharomyces cerevisiae or the cdc6-1 gene of Schizosaccharomyces pombe to the engineered S. cerevisiae TIY20 strain and S. pombe KT97 strain, respectively. TIY20, which is deficient in the outer chain of mannan due to the disruption of three genes (och1Delta, mnn1 Delta, mnn4 Delta), and KT97, which is an och1 disruptant, are impractical as hosts for the production of therapeutic glycoproteins since they show a temperature-sensitive (ts) phenotype, a growth defect phenotype, and decreased protein productivity. We successfully isolated YAB mutants that alleviated the growth defect of the TIY20 strain. Surprisingly, these mutants generally secreted foreign proteins better than the wild-type strain. Furthermore, we successfully isolated YPAB mutants that alleviated the growth defect of the KT97 strain, too. The development of these new mutants by the combination of genetic engineering of yeast and this mutagenesis technique are major breakthroughs for the production of therapeutic glycoproteins in engineered yeast cells.

Localization of type I myosin and F-actin to the leading edge region of the forespore membrane in Schizosaccharomyces pombe.

Myo1, a heavy chain of type I myosin of the fission yeast Schizosaccharomyces pombe, is essential for sporulation. Here we have analyzed the expression, localization and cellular function of the type I myosin light chain calmodulin, Cam2, encoded by cam2(+). Transcription of cam2(+) was constitutive and markedly enhanced in meiosis. The cam2 null mutant was viable and completed sporulation normally at 28 degrees C, but formed four-spored asci poorly at 34 degrees C. In those sporulation-defective cells, the forespore membrane was formed abnormally. A Cam2-GFP fusion protein accumulated at the cell poles in interphase cells and at the medial septation site in postmitotic cells, colocalizing with Myo1 and F-actin patches. During the mating process, a single Cam2-GFP dot was detected at the tip of the mating projection. During meiosis-I, the Cam2-GFP dots dispersed into the cell periphery and the cytoplasm. At metaphase-II, intense Cam2-GFP signals appeared near Meu14 rings which were formed at the leading edge of expanding forespore membranes. This localization of Cam2 was dependent upon Myo1; and sporulation defect of cam2Delta at 34 degrees C was alleviated by overexpressing Myo1DeltaIQ. These results suggest a close relationship between Cam2 and Myo1. In addition, both F-actin and Myo1 localized with Cam2 in the leading edge region. In summary, type I myosin and F-actin accumulate at the leading edge area of the forespore membrane and may play a pivotal role in its assembly.

Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae.

Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.