Protein transport in the permeabilized cell of Schizosaccharomyces pombe.

We reconstituted a protein translocation-transport system composed of permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe and the precursor of alpha sex pheromone, prepro-alpha-factor of the budding yeast Saccharomyces cerevisiae. We found that P-cells prepared from the spheroplasts formed in 0.7M KCl as an osmotic stabilizer had the activity to transport pro-alpha-factor to the Golgi apparatus. Electron microscopic observations showed that membranes were preserved more intact in the P-cells prepared from the spheroplasts formed in 0.7M KCl than in 0.7M sorbitol. A glycoprotein of S. pombe contains galactose residues, and we detected incorporation of radiolabeled galactose residues into the anti-prepro-alpha-factor immunoprecipitable fractions in this S. pombe system, but not in the S. cerevisiae system. This paper reports that a heterologous system of in vitro protein transport was performed, and prepro-alpha-factor has the signals necessary for early steps of the transport in S. pombe.     

Malate transport in Schizosaccharomyces pombe.

The transport of malate was studied in a Schizosaccharomyces pombe wild-type strain and in mutant strains unable to utilize malic acid. Two groups of such mutants, i.e., malic enzyme-deficient and malate transport-defective mutants, were differentiated by a 14C-labeled L-malate transport assay and by starch gel electrophoresis followed by activity staining for malic enzyme (malate dehydrogenase [oxaloacetate decarboxylating] [NAD+]; 1.1.1.38) and malate dehydrogenase (1.1.1.37). Transport of malate in S. pombe was constitutive and strongly inhibited by inhibitors of oxidative phosphorylation and of the formulation of proton gradients. Transport was a saturable function of the malate concentration. The apparent Km and Vmax values for transport by the parent were 3.7 mM and 40 nmol/min per mg of protein, respectively, while those of the malic enzyme-deficient mutant were 5.7 mM and 33 nmol/min per mg of protein, respectively. Malate transport was pH and temperature dependent. The specificity of transport was studied with various substrates, including mono- and dicarboxylic acids, and the possibility of a common transport system for dicarboxylic acids is discussed.     

Conjugation-induced lysis of Schizosaccharomyces pombe.

About 15% of the conjugating cells of Schizosaccharomyces pombe were observed to lyse spontaneously during the conjugation process. Lysis occurred at the site of union.     

Radiation resistance in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe serves as an excellent alternative and complementary model system for the analysis of genes and gene products involved in DNA repair. This brief review outlines the advantages of S. pombe and describes the radiation-sensitive mutants available for the analysis of DNA repair and recombination mechanisms in this organism. The progress in the cloning and characterization of representative genes is also described.     

Inhibition of Protein Translocation in Permeabilized Cells of Schizosaccharomyces pombe by Puromycin

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

PCR-mediated direct gene disruption in Schizosaccharomyces pombe.

We have examined the feasibility and efficiency of PCR-mediated direct gene disruptions in the fission yeast Schizosaccharomyces pombe. In the present study, the S.pombe ura4+ gene was amplified by PCR with oligonucleotides that had short flanking regions ( approximately 40 bp) to the target gene. Using this purified PCR product we were able to disrupt genes in an S. pombe strain bearing aura4 deletion, with an efficiency ranging between 1 and 3% among selected transformants. The results indicated that despite S.pombe's preference for non-homologous or illegitimate recombination, even very short stretches of homologous regions could be used to target genes at a defined frequency in this organism. The successful disruption of four independent genes (sts1+, gcs1+, gsh2+and hmt1+) by this method further demonstrates that, despite the relatively low efficiency, the method is very feasible, and it's simplicity, especially when coupled to phenotype-based screening, should greatly facilitate disruption of genes in S.pombe.     

Pre-mRNA splicing mutants of Schizosaccharomyces pombe.

A collection of temperature sensitive (ts-) mutants was prepared by chemical mutagenesis of a wild type Schizosaccharomyces pombe strain. To screen the ts- mutants for pre-mRNA splicing defects, an oligodeoxynucleotide that recognizes one of the introns of the beta-tubulin pre-mRNA was used as a probe in a Northern blot assay to detect accumulation of intron sequences. This screening procedure identified three pre-mRNA splicing mutants from 100 ts- strains. The three mutants are defective in an early step of the pre-mRNA splicing reaction; none accumulate intermediates. The precursors that accumulate at 37 degrees C are polyadenylated. Analysis of the splicing of another pre-mRNA showed that the mutations are not specific for beta-tubulin. The total RNA pattern in the three splicing mutants appears to be normal. In addition, the amounts of the spliceosomal snRNAs are not drastically changed compared to the wild type and splicing of pre-tRNAs is not blocked. Genetic analyses demonstrate that all three splicing mutations are tightly linked to the ts- growth defects and are recessive. Crosses among the mutants place them in three complementation groups. The mutants have been named prp1, prp2 and prp3.     

A mechanosensitive ion channel in Schizosaccharomyces pombe.

Protoplast protuberances (blebs) of Schizosaccharomyces pombe were examined using the patch-clamp technique. In addition to several voltage-gated ion channels, we encountered the activities of a mechanosensitive ion channel with a conductance of 180 pS. Microscopic currents of one or two units were observed in some excised patches and ensemble currents of several tens of units were observed in all blebs examined in whole-bleb configuration. This channel opens at pressures of cm Hg applied to whole blebs and it passes cations, including Ca2+. It is inactivated by membrane depolarizations and blocked by Gd3+. We discuss the possible functions of such a channel, including its activation upon cell cycle dependent cytoskeletal reorganizations.     

Human p53 inhibits growth in Schizosaccharomyces pombe.

Overexpression of wild-type p53 in mammalian cells blocks growth. We show here that the overexpression of wild-type human p53 in the fission yeast Schizosaccharomyces pombe also blocks growth, whereas the overexpression of mutant forms of p53 does not. The p53 polypeptide is located in the nucleus and is phosphorylated at both the cdc2 site and the casein kinase II site in S. pombe. A new dominant mutation of p53, resulting in the change of a cysteine to an arginine at amino acid residue 141, was identified. The results presented here demonstrate that S. pombe could provide a simple system for studying the mechanism of action of human p53.     

Checkpoint controls in Schizosaccharomyces pombe: rad1.

'Checkpoint' controls ensure that the events of the cell cycle are completed in an orderly fashion. For example, such controls delay mitosis until DNA synthesis and repair of radiation-induced DNA damage are complete. The rad series of radiosensitive fission yeast mutants was examined to identify strains deficient for the DNA damage-responsive checkpoint control. Five were identified. A characterization of one (rad1-1) and the wild-type is presented. The rad1-1 mutant does not arrest after irradiation, is sensitive to killing by radiation and is not arrested by hydroxyurea, and thus is also deficient for the DNA synthesis-responsive checkpoint control. The radiosensitivity of the rad1-1 mutant was greatly reduced when irradiated and maintained for 6 h in a non-dividing (density inhibited) state, demonstrating that rad1-1 is repair proficient and radiosensitive only through failure to delay. The checkpoint controls for which rad1 is required appear to regulate G2-M progression through the activity of cdc2, here implicated in this role by the coincidence of the radiation transition point and the cdc2 execution point.     

UV-induced replicating instability in Schizosaccharomyces pombe

A pigmented adenine-requiring strain of S. pombe has been used to study some aspects of the UV-induced replicating instabilities in this yeast. The effect of the repair capacity of a cell on the frequency of such instabilities has been investigated by using three different radiation-sensitive mutants and by studying the influence of caffeine on the frequency of secondary mosaics. Data showed that UV-induced replicating instabilities are not influenced by changes in the repair capacity of the treated cell. Since two of the sensitive mutants used are known to have high spontaneous mutation rates and the third one shows reduced UV-induced mutability, the induction of replicating instabilities differs in this respect both from the induction of spontaneous and UV-induced mutations.