Yeast sequencing reports. Cloning and DNA sequence of a Kluyveromyces lactis ERD1 homologue

The ERD1 gene product is required for the correct localization of soluble proteins that normally reside in the endoplasmic reticulum (ER). Cell lacking ERD1 secrete resident ER proteins and, in addition, exhibit defects in the processing of glycoproteins. Here, the molecular characterization of the Kluyveromyces lactis ERD1 homologue is described. A comparison of the predicted sequences of the Saccharomyces cerevisiae and K. lactis Erd1 proteins indicates that they are about 30% identical and 50% similar in sequence. Despite low sequence identity, these proteins are predicted to be conserved structurally. Furthermore, the K. lactis protein can functionally complement an S. cerevisiae mutant containing a deletion of the entire ERD1 gene, indicating these two proteins are functional homologues. The GenBank data library Accession Number for the DNA sequence reported in this paper is UO4714.     

The SPL1 tRNA splicing gene of Candida maltosa and Candida albicans

The tRNA splicing gene SPL1-1 has been cloned and sequenced in Saccharomyces cerevisiae (Kolman and Soll, 1993). Sequence adjacent to the LEU2 gene in Candida maltosa showed some homology to the SPL1-1 gene of S. cerevisiae. This work describes the sequencing of the SPL1 tRNA splicing genes from C. maltosa and C. albicans and the analysis of these genes. Comparison of these sequences and the relationship observed between the LEU2 and SPL1 genes in these yeasts suggests that there may be some synteny amongst various species of yeasts. The coding region of the C. maltosa SPL1 region described in this work differs from previously described partial sequences in that it is a complete uninterrupted open reading frame. Two strains of C. maltosa were each shown to contain different alleles, one uninterrupted open reading frame and one disrupted open reading frame. The sequences have been deposited in the GenBank/EMBL data libraries under Accession Numbers X72940, AF000115, AF000116, AF000117, AF000118, AF000119 and AF000120. © 1998 John Wiley & Sons, Ltd.     

The GDI1 genes from Kluyveromyces lactis and Pichia pastoris: cloning and functional expression in Saccharomyces cerevisiae

The nucleotide sequences of 2.8 kb and 2.9 kb fragments containing the Kluyveromyces lactis and Pichia pastoris GDI1 genes, respectively, were determined. K. lactis GDI1 was found during sequencing of a genomic library clone, whereas the P. pastoris GDI1 was obtained from a genomic library by complementing a Saccharomyces cerevisiae sec19‐1 mutant strain. The sequenced DNA fragments contain open reading frames of 1338 bp (K.lactis) and 1344 bp (P. pastoris), coding for polypeptides of 445 and 447 residues, respectively. Both sequences fully complement the S. cerevisiae sec19‐1 mutation. They have high degrees of homology with known GDP dissociation inhibitors from yeast species and other eukaryotes. The GenBank Accession Nos of the sequences are AF255332 (K.lactis GDI1) and AY007574 (P. pastoris GDI1). Copyright © 2001 John Wiley & Sons, Ltd.     

Kluyveromyces marxianus small DNA fragments contain both autonomous replicative and centromeric elements that also function in Kluyveromyces lactis

Two fragments containing both an autonomous replicating sequence (ARS) and a centromere have been isolated and sequenced from the yeast Kluyveromyces marxianus. The ARS and centromeric core sequences are only 500 bp apart, but ARS activity could be separated from the centromeric sequences. Centromeric sequences are organized in a similar way to those of budding yeasts: two well-conserved elements: CDEI (5′ TCACGTG 3′) and CDEIII (5′ TNTTCCGAAAGTWAAA 3′), are separated by a 165 bp AT-rich (± 90%) CDEII element whose length is twice that of Saccharomyces cerevisiae CDEII but almost identical to that of K. lactis. The ARS-core consensus sequence (5′ TTTATTGTT 3′) is also similar to that of K. lactis. Both ARS and centromeric elements function in this strain, albeit inefficiently, but not in S. cerevisiae. A third ARS-containing fragment with a different organization has been isolated and sequenced. The nucleotide sequences of DNA fragments reported in this paper will appear in the EMB data library under the accession numbers: Z31562, Z31563, Z31564.     

Candida albicans CaPTC6 is a functional homologue for Saccharomyces cerevisiae ScPTC6 and encodes a type 2C protein phosphatase

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ions. There are seven PP2C genes, ScPTC1, ScPTC2, ScPTC3, ScPTC4, ScPTC5, ScPTC6 and ScPTC7, in Saccharomyces cerevisiae. PTC6 is highly conserved in pathogenic and nonpathogenic yeasts. In the current study we have demonstrated that the Candida albicans CaPTC6 gene could complement the functions of ScPTC6 in the rapamycin and caffeine sensitivities of S. cerevisiae cells, indicating that they are functional homologues. We have also demonstrated that the CaPTC6‐encoded protein is a typical PP2C enzyme and that CaPtc6p is localized in the mitochondrion of yeast‐form and hyphal cells. However, deletion of CaPTC6 neither affects cell and hyphal growth nor renders Candida cells sensitive to rapamycin and caffeine. Therefore, possibly with a functional redundancy to other mitochondrial phosphatases, CaPtc6p is likely to be involved in the regulation of a mitochondrial physiology. Copyright © 2009 John Wiley & Sons, Ltd.     

Sequence comparison of the ARG4 chromosomal regions from the two related yeasts,Saccharomyces cerevisiae and Saccharomyces douglasii

A 3·6 kb DNA fragment from Saccharomyces douglasii, containing the ARG4 gene, has been cloned, sequenced and compared to the corresponding region from Saccharomyces cerevisiae. The organization of this region is identical in both yeasts. It contains besides the ARG4 gene, another complete open reading frame (ORF) (YSD83) and a third incomplete one (DED81). The ARG4 and the YSD83 coding regions differ from their S. cerevisiae homologs by 8.1% and 12·5%, respectively, of base substitutions. The encoded proteins have evolved differently: amino acid replacements are significantly less frequent in Arg4 (2·8%) than in Ysc83 (12·4%) and most of the changes in Arg4 are conservative, which is not the case for Ysc83. The non-coding regions are less conserved, with small AT-rich insertions/deletions and 20% base substitutions. However, the level of divergence is smaller in the aligned sequences of these regions than in silent sites of the ORFs, probably revealing a higher degree of constraints. The Gcn4 binding site and the region where meiotic double-strand breaks occur, are fully conserved. The data confirm that these two yeasts are evolutionarily closely related and that comparisons of their sequences might reveal conserved protein and DNA domains not expected to be found in sequence comparisons between more diverged organisms.     

Effect of pure and mixed cultures of the main wine yeast species on grape must fermentations

Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. We have analysed how mixed inocula of the main non-Saccharomyces yeasts and S. cerevisiae affect fermentation performance, nitrogen consumption and volatile compound production in a natural Macabeo grape must. Sterile must was fermented in triplicates and under the following six conditions: three pure cultures of S. cerevisiae, Hanseniaspora uvarum and Candida zemplinina and the mixtures of H. uvarum:S. cerevisiae (90:10), C. zemplinina:S. cerevisiae (90:10) and H. uvarum:C. zemplinina:S. cerevisiae (45:45:10). The presence of non-Saccharomyces yeasts slowed down the fermentations and produced higher levels of glycerol and acetic acid. Only the pure H. uvarum fermentations were unable to finish. Mixed fermentations consumed more of the available amino acids and were more complex and thus better able to synthesise volatile compounds. However, the amount of acetic acid was well above the admissible levels and compromises the immediate application of mixed cultures.     

Functional characteristics of Svl3 and Pam1 that are required for proper cell wall formation in yeast cells

In the budding yeast Saccharomyces cerevisiae, Svl3 and Pam1 proteins work as functional homologues. Loss of their function causes increased levels of chitin deposition in the cell wall and temperature sensitivity, suggesting their involvement in cell wall formation. We found that the N- and C-termini of these proteins have distinctive and critical functions. They contain an N-terminal part that has a probable 2-dehydropantoate 2-reductase domain. In Svl3, this part can be replaced with the yeast 2-dehydropantoate 2-reductase, Pan5, suggesting that Svl3 and its homologues may be able to mediate 2-dehydropantoate 2-reductase function. On the other hand, Svl3 is recruited to the bud tip and bud neck via multiple localization signals in the C-terminal part. One of such signals is the lysine-rich region located in the C-terminal end. The function and localization of Svl3 are significantly disrupted by the loss of this lysine-rich region; however, its localization is not completely abolished by the mutation because another localization signal enables appropriate transport. Svl3 and Pam1 orthologues are found in cells across fungal species. The Svl3 orthologues of Candida glabrata can complement the loss of Svl3 and Pam1 in S. cerevisiae. C. glabrata cells lacking the SVL3 and PAM1 orthologue genes exhibit phenotypes similar to those observed in svl3∆pam1∆ S. cerevisiae cells. Thus, Svl3 homologues may be generally required for the assembly of the cell wall in fungal cells.     

Mdm31 protein mediates sensitivity to potassium ionophores but does not regulate mitochondrial morphology or phospholipid trafficking in Schizosaccharomyces pombe

Mdm31p is an inner mitochondrial membrane (IMM) protein with unknown function in Saccharomyces cerevisiae. Mutants lacking Mdm31p contain only a few giant spherical mitochondria with disorganized internal structure, altered phospholipid composition and disturbed ion homeostasis, accompanied by increased resistance to the electroneutral K⁺/H⁺ ionophore nigericin. These phenotypes are interpreted as resulting from diverse roles of Mdm31p, presumably in linking mitochondrial DNA (mtDNA) to the machinery involved in segregation of mitochondria, in mediating cation transport across IMM and in phospholipid shuttling between mitochondrial membranes. To investigate which of the roles of Mdm31p are conserved in ascomycetous yeasts, we analysed the Mdm31p orthologue in Schizosaccharomyces pombe. Our results demonstrate that, similarly to its S. cerevisiae counterpart, SpMdm31 is a mitochondrial protein and its absence results in increased resistance to nigericin. However, in contrast to S. cerevisiae, Sz. pombe cells lacking SpMdm31 are also less sensitive to the electrogenic K⁺ ionophore valinomycin. Moreover, mitochondria of the fission yeast mdm31Δ mutant display no changes in morphology or phospholipid composition. Therefore, in terms of function, the two orthologous proteins appear to have considerably diverged between these two evolutionarily distant yeast species, possibly sharing only their participation in ion homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.     

Isolation and characterization of the Candida albicans SEC4 Gene

The SEC4 gene product is a major component of the protein secretion machinery. More specifically, it is believed to play a pivotal role in targeting and fusion of secretory vesicles to the plasma membrane. Its recently described implication with the Saccharomyces cerevisiae Rho3p, which is required for directing growing points during bud formation, has prompted us to investigate the role and function of Sec4p in the morphological changes of the yeast pathogen Candida albicans. We have therefore cloned the C. albicans SEC4 gene. It encodes a 210 amino acids long protein sharing up to 75% homology to the S. cerevisiae homolog, when conserved changes are allowed. Its RNA is constitutively expressed in C. albicans grown under various physiological conditions. We also show that it can functionally complement a S. cerevisiae sec4 thermosensitive mutant. The sequence of the C. albicans SEC4 gene has been deposited in GenBank under Accession Number AF017183. © 1998 John Wiley & Sons, Ltd.     

Analysis of TFIIH subunit through isolation of the gene from Schizosaccharomyces pombe corresponding to that of Saccharomyces cerevisiae SSL1, reveals the presence of conserved structural motifs

We isolated a Schizosaccharomyces pombe (Sz. pombe) gene encoding the counterpart of the TFIIH subunit Homo sapiens (H. sapiens) p44 and Saccharomyces cerevisiae (S. cerevisiae) SSL1, and we named this gene product p47. Contrary to the case of SSL1, which is an essential gene of S. cerevisiae, p47 is not essential for the viability of Sz. pombe. The deduced amino acid sequence revealed that this TFIIH subunit is highly conserved during evolution. Comparison of the primary structures revealed differences in the predicted positions of introns in the Caenorhabditis elegans (C. elegans) gene encoding the p47 counterpart found during the genome project. A charged cluster in the N-terminal region is present in the two yeasts. Two putative zinc-binding motifs, an extended C2H2 zinc finger with a 'C8 motif' and a second putative zinc-binding motif common to the two TFIIH subunits, were also found, the former being completely conserved. The latter motif consists of five cysteine residues and is also present in hp44, SSL1 and another TFIIH subunit, human p34 (hp34). Since one zinc atom can bind to four ligands in zinc-binding motifs, the conservation of cysteine residues was given attention. This motif is completely conserved in p47 homologues derived from the four species. As one cysteine residue is not conserved among the homologues of hp34, the consensus of this motif is concluded to be Cys X2-Cys-X(10,12)-Cys-X2-Cys. This nucleotide sequence has been deposited in the GenBank Data Library under Accession Number AF017646.     

Isolation and sequence analysis of a K. lactis chromosomal DNA element able to autonomously replicate in S. cerevisiae and K. lactis

We have undertaken a search for autonomously replicating (ARSs) from Kluyveromyces lactis chromosomal DNA able to sustain plasmid replication in K. lactis and in Saccharomyces cerevisiae. The discovery of such sequences might be interesting for the comparison of ARSs from different sources and possibly useful for the construction of multivalent vectors. HindIII fragments from K. lactis chromosomal DNA were inserted in the YIp5 plasmid (lacking an origin of replication) and the resulting chimaeric plasmids were selected for the ability to transform S. cerevisiae. Four plasmids were identified and further analysed. Two contained the same 1.8 kb K. lactis fragment and transformed both K. lactis and S. cerevisiae with the same efficiency and stability, whereas the third transformed only S. cerevisiae and the fourth transformed K. lactis with a higher efficiency than S. cerevisiae. A detailed study was performed on the 1.8 kb fragment which exhibited ARS function in both yeasts. The fragment was subcloned using different restriction enzymes and Bal31 exonuclease. Subclones were tested for ARS function. ARS activities in the two yeasts were localized in the same 100 bp region. Sequencing demonstrated the presence in this region of the dodecanucleotide 5'ATTTATTGTTTT3' differing from the ARS core consensus of S. cerevisiae only by a T insertion. A similar nucleotide sequence is present in the putative replication origin of the 2 mu-like plasmid pKD1 which stably replicates in K. lactis. Homologies with ARSs from S. cerevisiae were also found in the regions flanking the above-mentioned dodecanucleotide.     

Evolution of gene order and chromosome number in Saccharomyces,Kluyveromyces and related fungi

The extent to which the order of genes along chromosomes is conserved between Saccharomyces cerevisiae and related species was studied by analysing data from DNA sequence databases. As expected, the extent of gene order conservation decreases with increasing evolutionary distance. About 59% of adjacent gene pairs in Kluyveromyces lactis or K. marxianus are also adjacent in S. cerevisiae, and a further 16% of Kluyveromyces neighbours can be explained in terms of the inferred ancestral gene order in Saccharomyces prior to the occurrence of an ancient whole-genome duplication. Only 13% of Candida albicans linkages, and no Schizosaccharomyces pombe linkages, are conserved. Analysis of gene order arrangements, chromosome numbers, and ribosomal RNA sequences suggests that genome duplication occurred before the divergence of the four species in Saccharomyces sensu stricto (all of which have 16 chromosomes), but after this lineage had diverged from Saccharomyces kluyveri and the Kluyveromyces lactis/marxianus species assemblage. © 1998 John Wiley & Sons, Ltd.     

Role of O-acetylhomoserine sulfhydrylase in sulfur amino acid synthesis in various yeasts

Mutants defective in O-acetylhomoserine sulfhydrylase (OAH-SHLase) were obtained in five yeast strains representative of different yeast genera: Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe and Trichosporon cutaneum. In vitro, in all five strains, the enzyme also had O-acetylserine (OAS) sulfhydrylase activity so it is a ‘bifunctional’ OAH/OAS-SHLase (Yamagata, 1989). The enzyme was only found to be essential in S. cerevisiae (OAH SHLase-negative mutants are auxotrophs). Its impairment in K. lactis caused a slower growth rate and a decrease of the sulfur amino acid pool. In T. cutaneum only the pool was affected whereas in Y. lipolytica and S. pombe the lesion caused no change in the growth rate nor in the pool. In all strains where OAH SHLase-negative mutants were prototrophs, a monofunctional OAS sulfhydrylase was detected. The results indicate that OAH SHLase may play different physiological roles in various yeasts.     

Comparison of expression systems in the yeasts Saccharomyces cerevisiae,Hansenula polymorpha,Klyveromyces lactis,Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1α and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF gene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509. © 1998 John Wiley & Sons, Ltd.     

Differentiation between fermenting and spoilage yeasts in wine by total free fatty acid analysis

Differentiation between fermenting and spoilage yeasts in wine was estimated by cellular fatty acid profiles. Forty-two strains of yeasts representing 17 genera were grown on a defined liquid medium for 48 h at 25°C in a rotary shaker. After saponification of yeast cells, free fatty acid extracts were analysed by gas chromatography. Multivariate analysis was performed by Principal Components Analysis (PCA) to define clusters of fatty acids and yeasts. Strains were characterised especially by long-chain fatty acids, palmitic (C16) to linolenic (C18:3) acid under aerobic culture. Nevertheless, most of Saccharomyces cerevisiae and also Dekkera bruxellensis (former names D intermedia and Brettanomyces lumbicus) synthesized medium-chain fatty acids, octanoic (C8) acid to dodecanoic (C12) acid. With this method it was possible to differentiate fermenting grape yeasts such as S cerevisiae from spoilage yeasts on the basis of the absence of linoleic (C18:2) and linolenic (C18:3) acids. However, the method seemed unreliable for the identification of strains, more particularly those of species of S cerevisiae.     

The Essential Schizosaccharomyces pombe gpi1+ Gene Complements a Bakers' Yeast GPI Anchoring Mutant and is Required for Efficient Cell Separation

The Schizosaccharomyces pombe gpi1⁺ gene was cloned by complementation of the Saccharomyces cerevisiae gpi1 mutant, which has temperature-sensitive defects in growth and glycosyl phosphatidylinositol (GPI) membrane anchoring of protein, and which is defective in vitro in the first step in GPI anchor assembly, the formation of N -acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). S. pombe gpi1⁺ encodes a protein with 29% identity to amino acids 87–609 of the S. cerevisiae protein, and is the functional homolog of the S. cerevisiae Gpi1 protein, for it restores [³H]inositol-labelling of protein and in vitro GlcNAc-PI synthetic activity to both S. cerevisiae gpi1 and gpi1::URA3 cells. Disruption of gpi1⁺ is lethal. Haploid Δgpi1⁺::his7⁺ spores germinate, but proceed through no more than three rounds of cell division, many cells ceasing growth as binucleate, septate cells with thickened septa. These results indicate that GPI synthesis is an essential function in fission yeast, and suggest that GPI anchoring is also required for completion of cytokinesis. The nucleotide sequence reported will appear in the GenBank Nucleotide Sequence database under the Accession Number U77355.©1997 John Wiley & Sons, Ltd.     

Contribution of wild yeasts to the formation of volatile compounds in inoculated wine fermentations

The aim of this work was to study the contribution of wild yeasts to the volatile composition of wine in inoculated fermentations. To do so, Parellada must, sterilized and inoculated with Saccharomyces cerevisiae strain Na33 (pure inoculated fermentation), inoculated Parellada must (mixed inoculated fermentation) and Parellada must that fermented with its wild yeasts (control fermentation) were used. From the results obtained in the pure inoculated fermentation it can be seen that S. cerevisiae produced appreciable quantities of isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate. However, the wild yeasts also contributed to the synthesis of esters since the total concentration of these substances was higher in the mixed inoculated fermentation than in the pure inoculated fermentation. 2-Phenylethyl acetate was only synthesized by wild yeasts when they did not compete with S. cerevisiae. The concentration of total alcohols was similar in the three samples; the important production of isobutanol and 2-phenylethanol in the control fermentation is noteworthy. As regards the acids, the greatest concentration corresponded to the mixed inoculated fermentation. The wild yeasts contributed to the synthesis of these compounds to a significant extent and S. cerevisiae synthesized appreciable amounts of short-chain fatty acids.     

Secretion of Mouse α-Amylase from Kluyveromyces lactis

We constructed two mouse α-amylase secretion vectors for Kluyveromyces lactis using the well-characterized signal sequence of the pGKL 128 kDa killer precursor protein. Both PHO5 and PGK expression cassettes from Saccharomyces cerevisiae directed the expression of mouse α-amylase in YPD medium at a similar level of efficiency. K. lactis transformants secreted glycosylated and non-glycosylated α-amylase into the culture medium and both species were enzymatically active. The K. lactis/S. cerevisiae shuttle secretion vector pMI6 was constructed, and K. lactis MD2/1(pMI6) secreted about four-fold more α-amylase than S. cerevisiae YNN27 harboring the same plasmid, indicating that K. lactis is an efficient host cell for the secretion and production of recombinant proteins. © 1997 John Wiley & Sons, Ltd.