Eukaryotic DNA polymerases δ and ε, 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 δ variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38°C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40°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°C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA δ polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.

ABSTRACT. 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°C, but formed four-spored asci poorly at 34°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 cam2∆ at 34°C was alleviated by overexpressing Myo1∆IQ. 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.

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.

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.

Fission yeast sporulation seems to accompany a dynamic alteration of membrane traffic pathways in which the destination of secretory vesicles changes from the plasma membrane to the developing spore membrane. Evidence shows that endocytosis is responsible for this alteration in traffic pathways via the relocalization of syntaxin 1.

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