The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids

The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein is similar to models proposed for other integral mambrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1·5 kb. The mae1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5′ to the MFm1 gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid. The sequence of the S. pombe mae1 gene is available in GenBank under Accession Number U21002.     

A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast

We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-α-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A–Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-α-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism.     

Properties and Heterologous Expression of the Glucose Transporter GHT1 from Schizosaccharomyces pombe

Genomic DNA of the Schizosaccharomyces pombe glucose transporter, GHT1, was obtained by complementation of the glucose transport deficient Sz. pombe strain YGS-5. Here we describe the GHT1 gene that encodes a protein of 565 amino acids with a corresponding molecular mass of 62·5 kDa. This eukaryotic glucose transporter contains 12 putative transmembrane segments and is homologous to the HXT multigene family of S. cerevisiae with several amino acid motifs of this sugar transporter family. It is also homologous to other sugar carriers from human, mouse and Escherichia coli. The function of the Ght1 protein as a glucose transporter was proved both by homologous and heterologous expression in the Sz. pombe mutant YGS-5 and in the S. cerevisiae hxt mutant RE700A, respectively. Both transformed yeast strains transported d -glucose with substrate specificity similar to that in Sz. pombe wild-type cells. Moreover, the cells of the two transformed yeast strains accumulated 2-deoxy-d -glucose, a non-metabolizable d -glucose analogue, with an efficiency similar to Sz. pombe wild-type cells. The ability of the S. cerevisiae mutant RE700A to accumulate 2DG in an Δμdependent manner after transformation with GHT1 provides evidence that the Sz. pombe transporter catalyses an energy-dependent uptake of glucose. The sequence of GHT1 was deposited at EMBL, Outstation EBI, Accession Number X91218. ©1997 John Wiley & Sons, Ltd.     

X. Yeast sequencing reports. Sequence analysis of a 40·2 kb DNA fragment located near the left telomere of yeast chromosome X

We have sequenced on both strands a 40,257 bp fragment located near the left telomere of chromosome X of Saccharomyces cerevisiae. The sequenced segment contains 21 open reading frames (ORFs) at least 100 amino acids long. Five of the ORFs correspond to known amino acid sequences: two hypothetical proteins in the subtelomeric Y′ repeat region of 65·4 and 12·8 KDa, the cytochrome B pre-mRNA processing CBP1 protein, the mitochondrial nuclease NUC1 and the CRT1 protein. Of the 16 remaining ORFs, eight show highest homologies with the S. cerevisiae hexose transporters family (two ORFs), the yeast α-glucosidase (two ORFs), the yeast PEP1 precursor, the Escherichia coli galactoside O-acetyltransferase, the S. cerevisiae 137·7 KDa protein located in the Y′ region and a protein of unknown function of Schizosaccharomyces pombe. Finally, eight of the ORFs exhibit no significant similarity with any amino acid sequences described in data banks. DNA sequence comparison has revealed the presence of different repeated elements characteristic of yeast chromosome ends. Disruption studies have been performed on two ORFs encoding putative proteins of unknown function. The sequence has been entered in the EMBL Data Library under Accession Number Z34098.     

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

Analysis of the DNA Sequence of a 34 038 bp Region on the Left Arm of Yeast Chromosome XV

We report the DNA sequence of a 34 038 bp segment of Saccharomyces cerevisiae chromosome XV. Subsequent analysis revealed 20 open reading frames (ORFs) longer than 300 bp and two tRNA genes. Five ORFs correspond to genes previously identified in S. cerevisiae, including RPLA2, PRE6, MSE1, IFM1 and SCM2 (TAT2, TAP2, LTG3). Two putative proteins share considerable homology with other proteins in the current data libraries. ORF O2145 shows 41·2% identity with the glycophospholipid-anchored surface glycoprotein Gas1p of S. cerevisiae and ORF O2197 has 53·2% identity to chromosome segregation protein Dis3p of Schizosaccharomyces pombe. Accession Numbers for these sequences are provided in Table 1.©1997 John Wiley & Sons, Ltd.     

Characterization of the Saccharomyces cerevisiae cdc42-1ts Allele and New Temperature-Conditional-Lethal cdc42 Alleles

Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton. The CDC42 gene was first identified by the temperature-sensitive cell-division-cycle mutant cdc42-1^ts in Saccharomyces cerevisiae. We have determined the DNA and predicted amino-acid sequence of the cdc42-1^ts allele and identified multiple mutations in the coding region and 5′ promoter region, thereby limiting its usefulness in genetic screens. Therefore, we generated additional temperature-conditional-lethal alleles in highly conserved amino-acid residues of both S. cerevisiae and Schizosaccharomyces pombe Cdc42p. The cdc42^W97R temperature-sensitive allele in S. cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1^ts mutant. However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23°C. These phenotypes suggest that Cdc42p functions in bud-site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process. In S. pombe, the cdc42^W97R mutant displayed a cold-sensitive, loss-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions. In addition, cdc42^T58A and cdc42^S71P mutants showed a temperature-sensitive loss-of-function phenotype when expressed in S. pombe; these mutants did not display a conditional phenotype when expressed in S. cerevisiae. These new conditional-lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts. © 1997 John Wiley & Sons, Ltd.     

The translation initiation factor eIF4A from Schizosaccharomyces pombe is closely related to its mammalian counterpart

We have isolated a cDNA clone encoding eIF4A from Schizosaccharomyces pombe. The deduced protein sequence is similar in length and sequence to other eIF4A proteins and exhibits highest similarity with the mammalian eIF4A protein. Hybridization with genomic DNA reveals two eIF4A genes located on two different chromosomes. This sequence has been deposited in the EMBL Data Library under Accession Number X80796.     

Review: The Cct eukaryotic chaperonin subunits of Saccharomyces cerevisiae and other yeasts

All eight of the CCT1-CCT8 genes encoding the subunits of the Cct chaperonin complex in Saccharomyces cerevisiae have been identified, including three that were uncovered by the systematic sequencing of the yeast genome. Although most of the properties of the eukaryotic Cct chaperonin have been elucidated with mammalian systems in vitro, studies with S. cerevisiae conditional mutants revealed that Cct is required for assembly of microtubules and actin in vivo. Cct subunits from the other yeasts, Candida albicans and Schizosaccharomyces pombe, also have been identified from partial and complete DNA sequencing of genes. Cct8p from C. albicans, the only other completely sequenced Cct protein from a fungal species other than S. cerevisiae, is 72% and 61% similar to the S. cerevisiae and mouse Cct8 proteins, respectively. The C. albicans CCT8 sequence has been assigned the Accession Number U37371 in the GenBank/EMBL database.     

The uracil permease of Schizosaccharomyces pombe: A representative of a family of 10 transmembrane helix transporter proteins of yeasts

The uracil permease gene of Schizosaccharomyces pombe was cloned and sequenced. The deduced protein sequence shares strong similarities with five open reading frames from Saccharomyces cerevisiae, namely the uracil permease encoded by the FUR4 gene, the allantoin permease encoded by DAL4, a putative uridine permease (YBL042C) and two unknown ORFs YOR071c and YLR237w. A topological model retaining ten transmembrane helices, based on predictions and on experimental data established for the uracil permease of S. cerevisiae by Galan and coworkers (1996), is discussed for the four closest proteins of this family of transporters. The sequence of the uracil permease gene of S. pombe has been deposited in the EMBL data bank under Accession Number X98696. © 1998 John Wiley & Sons, Ltd.     

Molecular cloning of homologs of RAS and RHO1 genes from Cryptococcus neoformans

We cloned and sequenced homologs of RAS(CnRAS) and RHO1(CnRHO1) genes from Cryptococcus neoformans. The proteins encoded by the CnRAS and CnRHO1 genes contained 216 and 197 amino acids, respectively. The deduced amino acid sequence of the CnRAS gene shared a high degree of sequence identity with the Ras proteins in other fungal species: Coprinus cinereus(76%), Lentinula edodes(74%), Saccharomyces cerevisiae RAS2(72%), and Schizosaccharomyces pombe(68%). The deduced amino acid sequence of the CnRHO1 gene shared a high degree of sequence identity with the Rho1 proteins in other fungal species: Candida albicans(78%), S. pombe(77%) and S. cerevisiae(76%). The deduced proteins contained GTP‐binding and GTP‐hydrolysis domains, and the prenylation site that are conserved among the small G protein superfamily. The synthetic peptides that contained the C‐terminal amino acid sequence of the CnRas and CnRho1 proteins were geranylgeranylated. The DDBJ/EMBL/GenBank Accession Numbers of the CnRAS and CnRHO1 genes are AB017640 and AB017639, respectively. Copyright © 1999 John Wiley & Sons, Ltd.     

Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle

The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.     

Isolation of a Schizosaccharomyces pombe Gene Which in High Copy Confers Resistance to the Nucleoside Analogue 5-Azacytidine

Treatment of Schizosaccharomyces pombe with the C5 DNA methyltransferase (C5Mtase) inhibitor 5-azacytidine (5-azaC) has previously been shown to induce G2 checkpoint-dependent cell cycle arrest. S. pombe strains defective in both the checkpoint control pathways and in DNA repair processes are sensitive to 5-azaC. Here we describe the isolation of azr1as a multi-copy suppressor of the 5-azaC sensitivity of G2 checkpoint and DNA repair-deficient strains. azr1⁺ encodes a putative 25 kDa protein with limited homology to a Saccharomyces cerevisiae open reading frame of unknown function. The azr1⁺ gene is not essential and the null mutant shows no alteration in either DNA repair or checkpoint properties. We also report the sequence of the putative fission yeast cytidine deaminase gene, designated pcd1⁺, which lies immediately adjacent to azr1⁺ but which plays only a moderate role in suppression of 5-azaC sensitivity. These data have been deposited with EMBL nucleotide sequence database, Accession Number X98329. © 1997 John Wiley & Sons, Ltd.     

XIV. Yeast sequencing reports. The sequence of a 44 420 bp fragment located on the left arm of chromosome XIV from Saccharomyces cerevisiae

We have determined the complete nucleotide sequence of a 44 420 bp DNA fragment from chromosome XIV of Saccharomyces cerevisiae. The sequence data revealed 23 open reading frames (ORFs) larger than 300 bp, covering 73·5% of the sequence. The ORFs N2418, N2441, N2474 and N2480 correspond to previously sequenced S. cerevisiae genes coding respectively for the mitochondrial import protein Mas5, the nucleolar protein Nop2, the outer mitochondrial membrane porin Por1, the cytochrome c oxidase polypeptide VA precursor CoxA and the yeast protein tyrosine phosphatase Msg5. Translation products of three other ORFs N2406, N2411 and N2430 exhibit similarity to previously known S. cerevisiae proteins: the ribosomal protein YL9A, the protein Nca3 involved in the mitochondrial expression of subunits 6 and 8 of the ATP synthase and actin; in addition N2505 presents strong similarity to an ORF of chromosome IX. The predicted protein products of ORFs N2417 and N2403 present similarities with domains from proteins of other organisms: the Candida maltosa cycloheximide-resistance protein, the human interleukin enhancer-binding factor (ILF-2). The 12 remaining ORFs show no significant similarity to known proteins. In addition, we have detected a DNA region very similar to the yeast transposon Ty 1–15 of which insertion has disrupted a tRNA^Asp gene. The sequence has been deposited in the GenBank database with the Accession Number U12141.     

Single-read sequence tags of a limited number of genomic DNA fragments provide an inexpensive tool for comparative genome analysis

Single-read sequences from both ends of 415 3-kb average size genomic DNA fragments of Candida albicans were compared with the complete sequence data of Saccharomyces cerevisiae. Comparison at the protein level, translated DNA against protein sequences, revealed 138 sequence tags with clear similarity to S. cerevisiae proteins or open reading frames. One case of synteny was found for the open reading frames of RAD16 and LYS2, which are adjacent to each other in S. cerevisiae and C. albicans. © 1998 John Wiley & Sons, Ltd.     

Identification of a group-i intron within the 25S rDNA from the yeast Arxula adeninivorans

The 25S rDNA of the yeast Arxula adeninivorans LS3 has been cloned from a genomic library and sequenced. This DNA could be localized on chromosome 1 from A. adeninivorans and comprised 3790bp. The DNA sequence from this rDNA of the strain LS3 is very similar to the 25S rDNA of Candida albicans (91·7%), Saccharomyces cerevisiae (90·5%), Schizosaccharomyces pombe (83·8%) and Mucor racemosus (79·2%). Additionally a 411bp insertion could be localized within the 25S rDNA. This intervening sequence, which is devoid of any long open reading frame, is a group-IC intron as revealed from its site of insertion, predicted secondary structure, and its self-splicing capability. The Arxula intron is intermediate in structure and sequence between the ribosomal introns of Tetrahymena thermophila and C. albicans. The nucleotide sequence reported in this paper has been entered in the GenBank/EMBL data libraries and assigned Accession Number Z50840.     

Cloning and sequence of ADP-ribosylation factor 1 (ARF1) from Schizosaccharomyces pombe

A gene encoding a homologue of the ADP-ribosylation factor (ARF) family of small GTP binding proteins was cloned from a Schizosaccharomyces pombe cDNA library by a functional screen of suppressors of sensitivity to 3-aminotriazole in a gcn3 null strain of Saccharomyces cerevisiae. Two independent isolates each contained the full coding region of the ARF1 gene. The encoded SpARF1 protein has a predicted molecular weight of 20 618 and is 88% and 79% identical to human and S. cerevisiae ARF1 proteins, respectively. As independent isolates were obtained, this effect of the SpARF1 appears to be a real phenomenon, but cannot currently be easily understood within the context of the evidence for a role(s) for ARF proteins in the protein secretory pathway.