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-alpha-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.     

N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe

In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.     

Quality control in biosynthetic pathways of N-linked glycoproteins in the yeast endoplasmic reticulum

Summary The initial steps in N-glycosylation involve the synthesis of dolichol-linked Glc₃Man₉GlcNAc₂ oligosaccharides and the transfer of these oligosaccharides to nascent polypeptides. These processes take place at the membrane of the endoplasmic reticulum (ER) and are conserved among eukaryotes. Once transferred to the protein the N-linked oligosaccharides are immediately trimmed by glycosidases located in the ER. This review focuses on the N-linked glycosylation pathway in the ER ofSaccharomyces cerevisiae andSchizosaccharomyces pombe. In particular, we outline how yeast cells ensure that only completely assembled lipid-linked oligosaccharides are transferred to nascent polypeptides. We will discuss the oligosaccharide trimming of glycoproteins with respect to glycoprotein quality control and degradation, focusing on the two different quality control mechanisms ofS. cerevisiae andS. pombe.Abbreviations CPY carboxypeptidase Y - ER endoplasmic reticulum - LLO lipid-linked oligosaccharide - NLO protein-linked oligosaccharide - OTase oligosaccharyltransferase     

Expression of a VEGF-like Protein from Parapoxvirus ovis in the Yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe

We report on the expression of a VEGF-like protein encoded by Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We show that a lysine residue at amino acid position 2 (K2) is an important determinant for the stability of this protein in S. cerevisiae. Replacement of K2 by an arginine results in stabilization of the protein. This observation suggests that this lysine may be a target for ubiquitinylation, which is a prerequisite for proteasome-mediated protein degradation. Interestingly, in S. pombe the lysine (K2) has no influence on the stability of the protein. This result indicates that the two yeast species exhibit significant differences in their protein degradation pathways.     

Stability and refractoriness of the high catalase activity in the oxidative-stress-resistant fission yeastSchizosaccharomyces pombe

Effect of oxygen and metabolic substrates (glucose, ethanol) on the catalase activity of anaerobically grownSchizosaccharomyces pombe cells was assessed, and compared with that ofSaccharomyces cerevisiae in order to determine the catalase activity regulation inS. pombe. In contrast toS. cerevisiae, the total catalase activity of permeabilizedS. pombe anaerobically grown cells is higher than that found in aerobically grown cells, is stable and constant under all circumstances (i.e. it is not induced by oxygen and/or substrates), and only a negligible part (3–5%) of it is contributed byde novo protein synthesis during aeration with or without substrates. The patent catalase activity of intact cells rises 2-fold during 6-h aeration without substrate and 7–8-fold in the presence of glucose or ethanol. The increase is not inhibited by cycloheximide and is thus not due tode novo catalase synthesis, but may reflect enhanced transport of catalase to the cell surface or a permeabilization of the plasma membrane during the aeration.     

Reduction in the intracellular cAMP level triggers initiation of sexual development in fission yeast

Summary Schizosaccharomyces pombe initiates sexual development in response to nutritional starvation. The level of cAMP inS. pombe cells changed during the transition from exponential growth to stationary phase. It also changed in response to a shift from nitrogen-rich medium to nitrogen-free medium. A decrease of approximately 50% was observed in either case, suggesting thatS. pombe cells contain less cAMP when they initiate sexual development.S. pombe cells that expressed the catalytic domain ofSaccharomyces cerevisiae adenylyl cyclase from theS. pombe adh1 promoter contained 5 times as much cAMP as the wild type and could not initiate mating and meiosis. These observations, together with previous findings that exogenously added cAMP inhibits mating and meiosis and that cells with little cAMP are highly derepressed for sexual development, strongly suggest that cAMP functions as a key regulator of sexual development inS. pombe. Thepde1 gene, which encodes a protein homologous toS. cerevisiae cAMP phosphodiesterase I, was isolated as a multicopy suppressor of the sterility caused by a high cAMP level. Disruption ofpde1 madeS. pombe cells partially sterile and meiosis-deficient, indicating that this cAMP phosphodiesterase plays an important role in balancing the cAMP level in vivo.     

Intracellular pH inSchizosaccharomyces pombe — Comparison withSaccharomyces cerevisiae

We examined cytoplasmic pH regulation inSchizosaccharomyces pombe andSaccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 fromS. pombe was much slower than leakage fromC. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 forS. pombe and 6.6 forS. cerevisiae. We found that internal pH inS. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater inS. cerevisiae than inS. pombe. The plasma membrane H⁺-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC₅₀ of approximately 35 M in both species. Amiloride also inhibited internal alkalinisation with IC₅₀'s of 745 M forS. cerevisiae and 490 M forS. pombe.Abbreviations C.SNARF-1 carboxy-seminaphthorhodafluor-1 (-AM-acetoxy-methylester) - DES diethylstilbestrol - IC₅₀ apparent inhibitory constant - BCECF 2,7-bis-(carboxyethyl)-5(6)-carboxyfluorescein (-AM--pentaacetoxymethyl ester) - FDA fluorescein diacetate     

Nonsense suppression in Schizosaccharomyces pombe: the S. pombe Sup3-e tRNASerUGA gene is active in S. cerevisiae

Summary The gene encoding the efficient UGA suppressor sup3-e of Schizosaccharomyces pombe was isolated by in vivo transformation of Saccharomyces cerevisiae UGA mutants with S. pombe sup3-e DNA. DNA from a clone bank of EcoRI fragments from a S. pombe sup3-e strain in the hybrid yeast vector YRp17 was used to transform the S. cerevisiae multiple auxotroph his4-260 leu2-2 trp1-1 to prototrophy. Transformants were isolated at a low frequency; they lost the ability to grow in minimal medium after passaging in non-selective media. This suggested the presence of the suppressor gene on the non-integrative plasmid. Plasmid DNA, isolated from the transformed S. cerevisiae cells and subsequently amplified in E. coli, transformed S. cerevisiae his4-260 leu2-2 trp1-1 to prototrophy. In this way a 2.4 kb S. pombe DNA fragment carrying the sup3-e gene was isolated. Sequence analysis revealed the presence of two tRNA coding regions separated by a spacer of only seven nucleotides. The sup3-e tRNA _Ser ^UGA tRNA gene is followed by a sequence coding for the initiator tRNA^Met. The transformation results demonstrate that the cloned S. pombe UGA suppressor is active in S. cerevisiae UGA mutant strains.     

First identification of an amber nonsense mutation in Schizosaccharomyces pombe: major differences in the efficiency of homologous versus heterologous yeast suppressor tRNA genes

Summary In Saccharomyces cerevisiae ochre and opal, as well as amber mutations are known, whereas in the fission yeast Schizosaccharomyces pombe no amber alleles have been described. We have characterized trp1-566, an amber allele in the trp1 locus of S. pombe. The identification of trp1-566 as an amber allele is based on the following results: (a) The nonsense allele can be converted to an ochre allele by nitrosoguanidine mutagenesis. (b) trp1-566 is suppressed by a bona fide S. pombe amber suppressor tRNA, supSI. The supSI gene was obtained by primer-directed in vitro mutagenesis of a tRNASer from S. pombe. Unexpectedly, an S. cerevisiae amber suppressor tRNASer, supR21, transformed into S. pombe, failed to suppress trp1-566. Northern analysis of S. pombe transformants, containing supRL1 or S. cerevisiae tRNA^Leu or tRNA^Tyr genes reveals that these genes are not transcribed in the fission yeast. As an additional tool for the analysis of nonsense mutations in S. pombe, we obtained by nitrosoguanidine mutagenesis two unlinked amber suppressor alleles, sup13 and sup14, which act on trp1-566.     

The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae

The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium . This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.

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Isolation of the ribosomal protein gene S6 from Schizosaccharomyces pombe

Summary We screened a Schizosaccharomyces pombe genomic library using the ribosomal protein gene SI0 from Saccharomyces cerevisiae as a probe. Hybrid-selected translation of the positive clones revealed a ribosomal protein of S. pombe which is probably equivalent to the ribosomal protein SI0 from S. cerevisiae.     

Fluctuations during growth of the plasma membrane H(+)-ATPase activity of Saccharomyces cerevisiae and Schizosaccharomyces pombe

The plasma membrane H⁺-ATPase activity was determined under various growth conditions using the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe. Under early batch-growth conditions in a rich medium, the budding yeastS. cerevisiae ATPase specific activity increased 2-to 3-fold during exponential growth. During late exponential growth, a peak of ATPase activity, followed by a sudden decrease, was observed and termed “growth-arrest control”. The growth arrest phenomenon ofS. cerevisiae could not be related to the acidification of the culture medium or to glucose exhaustion in the medium or to variation of glucose activation of the H⁺-ATPase. Addition of ammonium to a proline minimum medium also stimulated transiently the ATPase activity ofS. cerevisiae. Specific activity of the fission yeastS. pombe ATPase did not show a similar profile and steadily increased to reach a plateau in stationary growth. Under synchronous mitotic growth conditions, the ATPase activity ofS. cerevisiae increased during the cell division cycle according to the “peak” type cycle, while that ofS. pombe was of the “step” type.     

Characterisation of PHSP1, a cDNA encoding a mitochondrial HSP70 fromPisum sativum

A pea cDNA clone,PHSP1, encoding a member of the HSP70 gene family has been isolated. DNA sequence analysis indicates that the protein encoded byPHSP1 is a homologue of the mitochondrial HSP70 proteins, SSP1 fromSchizosaccharomyces pombe and SSC1 fromS. cerevisiae. It contains an amino-terminal extension of 50 amino acids, rich in basic and hydroxyl amino acids, similar to other plant mitochondrial leader sequences. Western blot analysis indicates that the PHSP1 protein is associated only with mitochondria and not with any other sub-cellular organelle or cytoplasm. Further confirmation of its location within mitochondria was obtained fromin vitro protein translocation experiments into purifiedPisum sativum mitochondria. It was observed that the precursor protein was efficiently imported and that it is processed to produce a protein with anM _r of the anticipated size of the mature protein. Results are discussed with respect to the structure and function of the mitochondrial HSP70 protein.Abbreviations mtHSP70 mitochondrial HSP70 - ER endoplasmic reticulum - nt nucleotide - IgG immunoglobulin G - BiP immunoglobulin-binding protein - hsc heat shock cognate     

The RCK1 and RCK2 protein kinase genes from Saccharomyces cerevisiae suppress cell cycle checkpoint mutations in Schizosaccharomyces pombe

The protein kinase-encoding genes RCK1 and RCK2 from Saccharomyces cerevisiae have been identified as suppressors of Schizosaccharomyces pombe cell cycle checkpoint mutations. Upon expression of these genes, radiation resistance is partially restored in S. pombe mutants with checkpoint deficiencies, but not in mutants with DNA repair defects. Some checkpoint mutants are sensitive to the DNA synthesis inhibitor hydroxyurea, and this sensitivity is also suppressed by RCK1 and RCK2. The degree of suppression can be modulated by varying expression levels. Expression of RCK1 or RCK2 in S. pombe causes cell elongation and decelerated growth. Cells expressing these genes have a single nucleus and a 2n DNA content. We conclude that these genes act in S. pombe to prolong the G2 phase of the cell cycle.     

Conserved and unique features of the fission yeast core Atg1 complex

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature. Interestingly, S. pombe Atg17 binds S. cerevisiae Atg13, Atg29, and Atg31 in vitro, but it cannot complement the function of S. cerevisiae Atg17 in vivo. Furthermore, S. pombe Atg101 cannot substitute for the function of S. cerevisiae Atg29 and Atg31 in vivo. Collectively, our work generates new insights into the subunit organization and structural properties of an Atg101-containing Atg1/ULK complex.     

Size variation of rDNA clusters in the yeasts Saccharomyces cerevisiea and Schizosaccharomyces pombe

Summary The higher-order organization of rRNA genes was investigated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We used pulsed-field gel electrophoresis (PFGE) in combination with frequent cutter endonucleases having no recognition sites within rDNA repeating units to characterize tandem arrays of ribosomal genes in these two species. Large variations in rDNA cluster length were detected in various S. cerevisiae and S. pombe strains commonly used as PFGE molecular weight markers. This wide range of variability implies that the sizes currently assessed for chromosomes bearing rRNA genes in these organisms are unreliable since they may vary within strains by several hundreds of kilobase pairs, depending on the size of the tandem arrays of rRNA genes. Consequently, there is now a lack of reliable PFGE size standards between 1.6 Mb and 4.5 Mb, even when established yeast strains with calibrated chromosomes are used.     

Generation of expression vectors for high-throughput functional analysis of target genes in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, urw4 ⁺ and Saccharomyces cerevisiae LEU2 complementing leul. These vectors contain 3 different strengths of the inducible promoter nmtl, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3× HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.     

Yeast strains for concentrated substrates

Summary The screening of twenty yeast strains for ethanol productivity at high osmotic pressure at temperatures ranging from 32°C to 45°C is described. Shake flask fermentations of 30°, 40°, and 50° Bx cane molasses were performed. The effect of temperature on productivity at a non-inhibitory ethanol level is weakly pronounced. Most strains fermented poorly at 50° Bx molasses but two Schizosaccharomyces pombe and one commercial baker's yeast, Saccharomyces cerevisiae performed well at all concentrations of molasses. In an extended study with Schizosaccharomyces pombe (CBS 352) and Saccharomyces cerevisiae (SJAB, fresh yeast), simulating a continuous run it was shown that Schizosaccharomyces pombe was less sensitive to high DS than Saccharomyces cerevisiae. At 25% DS the productivity of Schizosaccharomyces pombe is almost twice that of Saccharomyces cerevisiae.