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C Gaillardin

Publications and source records attributed to C Gaillardin.

At least 91 records · Page 5Linked to original sources
PubMed1995

Sequencing analysis of a 24.7 kb fragment of yeast chromosome XIV identifies six known genes, a new member of the hexose transporter family and ten new open reading frames.

The DNA sequence of a 24.7 kb region covering the left arm of chromosome XIV from Saccharomyces cerevisiae was determined. This region contains 17 open reading frames (ORFs) which code for proteins of more than 100 amino acids. Five ORFs correspond to the KRE1, ATP11, DAL82, RFA2 and MCK1 loci, described previously. Two ORFs present high similarity to known proteins: NO345 with the hexose transporter family, and NO351 with the yeast chorismate mutase/prephenate dehydratase enzyme encoded by PHA2. Six ORFs show limited similarity with known proteins or some specific features: NO339 presents 11 potential transmembrane domains. NO343, which is internal to NO345, presents a putative signal sequence and a potential transmembrane domain. NO348 shows similarity with YCW2, TUP1 and SEC13. NO364 reveals a signature for a pyridoxal-phosphate attachment site. Finally, NO384 and NO388 present a biased amino acid composition, being rich in Asn or Glu/Lys/Arg, respectively. Four other ORFs (NO342, NO376, NO381 and NO397) show no similarity to proteins within the databases screened.

Amino Acid Sequence↗
PubMed1995

Sequencing analysis of a 15.4 kb fragment of yeast chromosome XIV identifies the RPD3, PAS8 and KRE1 loci, five new open reading frames.

The DNA sequence of a 15.4 kb region covering the left arm of chromosome XIV from Saccharomyces cerevisiae was determined. This region contains eight open reading frames (ORFs) which code for proteins of more than 100 amino acids. Three ORFs correspond to the RPD3, PAS8 and KRE1 loci, described previously. Three ORFs show limited homology with known proteins: NO330 with the recessive suppressor of secretory defect SAC1, NO325 with YCR094W identified during chromosome III sequencing; whereas NO315 presents a motif conserved in the dnaJ family. Two ORFs (NO320 and NO325) show no homology to known proteins within the databases screened, but NO320 corresponds to a serine-threonine-rich protein. The sequence has been entered in the EMBL data library under Accession Number Z46259.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of Rab-related genes in the yeast Yarrowia lipolytica. Analysis of RYL1, an essential gene encoding a SEC4 homologue.

Small GTP-binding proteins of the Rab family are involved in the vesicular traffic inside eukaryotic cells. A gene library from the yeast Yarrowia lipolytica was screened with an oligonucleotide deduced from a highly conserved sequence in the Rab family. Four different genes were isolated. One of them, RYL1, was shown to be essential for cell viability. RYL1p displayed a high similarity with and tight phylogenetic relationships to SEC4p. When placed under the control of the GAL10 promoter, RYL1 was able to specifically relieve the thermosensitivity of a sec4-8 mutant of Saccharomyces cerevisiae. Therefore, it is proposed that RYL1 is a functional homologue of the S. cerevisiae SEC4 gene and is involved in the fusion of secretory vesicles with the plasma membrane in the general protein secretion pathway.

Amino Acid Sequence↗
PubMed1995

Hyperthermostable mutants of Bacillus licheniformis alpha-amylase: multiple amino acid replacements and molecular modelling.

We have identified previously two critical positions for the thermostability of the highly thermostable alpha-amylase from Bacillus licheniformis. We have now introduced all 19 possible amino acid residues to these two positions, His133 and Ala209. The most favourable substitutions were to Ile and Val, respectively, which both increased the half-life of the enzyme at 80 degrees C by a factor of approximately 3. At both positions a stabilizing effect of hydrophobic residues was observed, although only in the case of position 133 could a clear correlation be drawn between the hydrophobicity of the inserted amino acid and the gain in protein stability. The construction of double mutants showed a cumulative effect of the most favourable and/or deleterious substitutions. Computer modelling was used to generate a 3-D structure of the wild-type protein and to model substitutions at position 209, which lies in the conserved (alpha/beta)8 barrel domain of alpha-amylase; Ala209 would be located at the beginning of the third helix of the barrel, in the bottom of a small cavity facing the fourth helix. The model suggests that replacement by, for example, a valine could fill this cavity and therefore increase intra- and interhelical compactness and hydrophobic interactions.

Amino Acid Sequence↗
PubMed1994

Development of a transformation system for the yeast Yamadazyma (Pichia) ohmeri.

This communication describes the development of genetic tools for the yeast Yamadazyma ohmeri. Nystatin enrichment proved highly effective for isolating various auxotrophic strains, which were classified by complementation analysis. Biosynthetic genes encoding known biochemical functions were isolated by polymerase chain reaction, including YoLEU2 and YoURA3 that were sequenced. Using these homologous genes as selective markers, DNA transformation was accomplished by electroporation. Transformation with pBR322-based plasmids, cut within the coding region of the homologous marker gene, yielded 20 to 50 stable transformants per microgram of DNA. In about 80% of the cases, integration of plasmid DNA sequence occurred by homologous recombination of a single plasmid into the chromosome. Excision of the plasmid permitted gene replacement, as illustrated by the substitution of a wild-type URA3 gene by an in vitro generated deletion. Sequences conferring extrachromosomal replication were isolated from Y. ohmeri DNA. Plasmids based on pBR322 carrying such an ARS and either selective markers transformed at 10(4)/microgram and were shown to replicate freely in Y. ohmeri at an approximate copy number of 40. Unexpectedly, we observed that BS-SKR derivatives carrying either YoLEU2 or YoURA3 but no Y. ohmeri ARS also replicated extrachromosomally. Linearization of transforming plasmids within regions homologous or not to chromosomal sequences stimulated transformation frequencies up to four-fold.

Amino Acid Sequence↗
PubMed1994

Multiple-copy integration in the yeast Yarrowia lipolytica.

Using an EcoRI-BglII fragment of the G unit of the rDNA of Y. lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA. These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E. coli. Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773). Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene. Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion. Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25-60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization. Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes. Multiple integrations were found both as tandem repeats and as dispersed copies. Plasmid copy number was stable, in both minimum and rich media, for strains containing less than ten copies per cells. However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.

Base Sequence↗
PubMed1994

LYC1 is the structural gene for lysine N-6-acetyl transferase in yeast.

In the yeast Yarrowia lipolytica, the LYC1 locus controls the first step of the lysine degradation pathway which is catalyzed by lysine N-6-acetyl transferase (LAT). This gene was cloned by complementation of the lyc1-100 mutation. Its position in the cloned insert was determined by conversion mapping and by complementation. The LYC1 gene encodes a 391 amino-acid polypeptide which has no homolog in protein databases. The required upstream region extends over 960 bp. When placed under the control of the GAL10 promoter in Saccharomyces cerevisiae, LYC1 drives the expression of lysine acetyl transferase activity, thus providing strong evidence that it is the structural gene encoding this enzyme.

Acetyltransferases↗
PubMed1994

A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form.

The SEC14SC gene encodes the phosphatidylinositol/phosphatidylcholine transfer protein (PI/PC-TP) of Saccharomyces cerevisiae. The SEC14SC gene product (SEC14pSC) is associated with the Golgi complex as a peripheral membrane protein and plays an essential role in stimulating Golgi secretory function. We report the characterization of SEC14YL, the structural gene for the PI/PC-TP of the dimorphic yeast Yarrowia lipolytica. SEC14YL encodes a primary translation product (SEC14YL) that is predicted to be a 497-residue polypeptide of which the amino-terminal 300 residues are highly homologous to the entire SEC14pSC, and the carboxyl-terminal 197 residues define a dispensible domain that is not homologous to any known protein. In a manner analogous to the case for SEC14pSC, SEC14pYL localizes to punctate cytoplasmic structures in Y. lipolytica that likely represent Golgi bodies. However, SEC14pYL is neither required for the viability of Y. lipolytica nor is it required for secretory pathway function in this organism. This nonessentiality of SEC14pYL for growth and secretion is probably not the consequence of a second PI/PC-TP activity in Y. lipolytica as cell-free lysates prepared from delta sec14YL strains are devoid of measurable PI/PC-TP activity in vitro. Phenotypic analyses demonstrate that SEC14pYL dysfunction results in the inability of Y. lipolytica to undergo the characteristic dimorphic transition from the yeast to the mycelial form that typifies this species. Rather, delta sec14YL mutants form aberrant pseudomycelial structures as cells enter stationary growth phase. The collective data indicate a role for SEC14pYL in promoting the differentiation of Y. lipolytica cells from yeast to mycelia, and demonstrate that PI/PC-TP function is utilized in diverse ways by different organisms.

Amino Acid Sequence↗
PubMed1994

Identification of new chromosomes of Saccharomyces bayanus using gene probes from S. cerevisiae.

The Saccharomyces cerevisiae genes HIS3 (chr. XV), LYS2 (chr. II), ARG4 (chr. VIII), MF alpha 1 (chr. XVI) and MEL1 (chr. II) and the S. pastorianus gene MELx were used for identification of the homeologous chromosomes in S. bayanus by using pulsed-field gel electrophoresis and Southern blot hybridization. The results showed differences in hybridization intensities and mobilities between the homeologous chromosomes of the two sibling yeasts S. cerevisiae and S. bayanus. The probes HIS3, LYS2, MEL1, and MF alpha 1 identified four new homeologous chromosomes of S. bayanus. The ARG4 probe confirmed the previously identified location of chr. VIII (NAUMOV et al. 1992b). Using the MF alpha 1 gene, another chromosome besides chr. XVI was identified in some S. bayanus strains, suggesting translocation of this sequence to the S. bayanus chromosome that normally migrates to the position of chr. XI of S. cerevisiae.

Chromosomes, Fungal↗
PubMed1994

Two upstream activation sequences control the expression of the XPR2 gene in the yeast Yarrowia lipolytica.

We have initiated a study of the promoter region of the alkaline extracellular protease gene (XPR2) from Yarrowia lipolytica to identify upstream sequences possibly involved in carbon, nitrogen, and peptone control of XPR2 expression. Deletion analysis showed that the TATA box and two major upstream activation sequences (UASs) were essential for promoter activity under conditions of repression or full induction. Within the distal UAS (UAS1), in vivo footprinting studies with dimethyl sulfate (DMS) identified two sequences similar to Saccharomyces cerevisiae GCN4 (-800 to -792)- and TUF/RAP1 (-790 to -778)-binding sites and two sequences which partially overlap a repeated sequence (-778 to -771 and -720 to -713) similar to the CAR1 upstream repression sequence of S. cerevisiae. Oligonucleotides carrying the TUF/RAP1-like-binding site and adjacent downstream nucleotides restored full transcriptional activity of a UAS1-deleted promoter. Within the proximal UAS (UAS2), a directly repeated decameric sequence (-146 to -137 and -136 to -127) was protected against DMS in vivo. Sequences identical to the ABF1-binding site of S. cerevisiae (-121 to -109) or similar to the GCN4-binding site (-113 to -105) were not clearly protected from DMS in vivo. An oligomer (-150 to -106) carrying these three sequences, inserted into a UAS2-deleted promoter, increased the transcriptional activity. The results from footprints under different physiological conditions suggested that protein binding to both UASs was constitutive. Deletion of both UASs greatly reduced XPR2 expression without abolishing its regulation. Our results strongly suggest that these UASs are targets for transcriptional factors required for assisting specific regulatory proteins.

Amino Acid Sequence↗
PubMed1992

Secretion of human blood coagulation factor XIIIa by the yeast Yarrowia lipolytica.

The industrial yeast, Yarrowia lipolytica, secretes high yields of an alkaline extracellular protease (AEP), which is synthesized as a preproprotein encoded by the XPR2 gene. We investigated the possibility of using this system for the secretion of human coagulation factor XIII subunit a (FXIIIa). This protein is naturally secreted in the plasma by an unknown, signal peptide-independent mechanism and has so far been found to be nonsecretable in yeast. We have designed six hybrid genes encoding fusion proteins between increasing portions of the AEP preprodomain and the precursor or mature forms of FXIIIa. All constructs directed translocation of the FXIIIa precursor into the endoplasmic reticulum. Transport of the translocated and core-glycosylated hybrid precursor to the Golgi apparatus appeared to be strongly rate limiting, and most of the precursors appeared to be partially proteolysed. One of these constructs directed the extracellular secretion of a low amount of hyperglycosylated FXIIIa. These results indicate that fusion to the yeast AEP signal peptide and dipeptide stretch allows FXIIIa to be translocated, albeit inefficiently, through the endoplasmic reticulum and to follow a classical secretory transit.

Amino Acid Sequence↗
PubMed1992

Intracellular transit of a yeast protease is rescued by trans-complementation with its prodomain.

The alkaline extracellular protease (AEP) of the yeast Yarrowia lipolytica is synthesized as a preproprotein. The precursor undergoes a complex maturation during its intracellular transit, successively involving signal peptide cleavage, dipeptidyl aminopeptidase processing, and cleavage at a dibasic site which results in the extracellular release of the active enzyme. It was previously shown that various deletions within the proregion affect the intracellular transit of the protease. Prodeleted precursors are translocated and have their signal sequences removed, but they accumulate in the secretion apparatus. We show here that the secretion of partially active proteins is restored when the prodomain is supplied in trans as an independent peptide. The secretion rescue and maturation processing that are reconstituted by the free propeptide do not reach wild type efficiency. The results of pulse-chase experiments indicate that a rate-limiting step occurs during the intracellular transit of the rescued precursors, before Kex2p proteolytic cleavage. This delayed maturation seems to be responsible for an overall slower release of the rescued polypeptides. Propeptide and AEP were secreted in equimolar amounts by both wild type and trans-complemented strains, but none could be detected in the supernatant when expressed alone. These experiments suggest that the prodomain of AEP initially acts as a crucial folding aid for the early secretory transit of the translocated precursor. They further suggest that the prodomain is also required for a second structural change of the AEP precursor during its activation.

Amino Acid Sequence↗
PubMed1992

A mutant of 7SL RNA in Yarrowia lipolytica affecting the synthesis of a secreted protein.

The yeast Yarrowia lipolytica contains two genes, SCR1 and SCR2, encoding a 7SL RNA associated with a signal recognition particle (SRP). To study 7SL RNA function in vivo we have systematically substituted the two conserved nucleotides G128 and A130 in loop 1 of SCR2-encoded 7SL RNA. All single mutations in either nucleotide have no effect. All double mutations are lethal except for one which results in temperature-sensitive growth. We have studied the synthesis and secretion of an alkaline extracellular protease (AEP) in both wild-type and temperature-sensitive mutant strains. Pulse-chase labeling and immunoprecipitation of this protein show that: 1) total protein synthesis is not affected in the mutant strain; 2) levels of AEP precursors in the mutant strain are 60% less than in the wild-type strain; 3) for both strains there is no accumulation of the AEP precursors in the cytoplasm; 4) the kinetics of secretion is similar. These results suggest that mutated SRP is deficient in membrane targeting but still performs translational arrest. This is consistent with the functions of SRP observed in vitro and represents the first demonstration of such roles in vivo.

Base Sequence↗
PubMed1992

Fission yeast and a plant have functional homologues of the Sar1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast.

Sec12p and Sar1p are required for the formation of transport vesicles generated from the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. Sec12p is an ER type II membrane protein that mediates the membrane attachment of the GTP-binding Sar1 protein. The SAR1 gene is a multi-copy suppressor of a thermosensitive sec12 mutation. In an attempt to identify functional homologues of Sec12p and Sar1p from other eukaryotic organisms, we screened cDNA expression libraries derived from the fission yeast Schizosaccharomyces pombe and from the plant Arabidopsis thaliana for complementation of the sec12ts mutation. Four individual cDNAs were isolated, two of which encode the S. pombe and A. thaliana homologues of Sar1p. The three Sar1 proteins are 67% identical on average. The two other cDNAs encode type II membrane proteins which were designated Stl1p for the S. pombe protein and Stl2p for the A. thaliana protein (Stl stands for Sec12p-like). Both proteins have NH2-terminal cytoplasmic domains which resemble that of Sec12p: they are similar in size and present a significant degree of amino acid identity with the cytoplasmic domain of Sec12p. In contrast, the lumenal domains of Sec12p, Stl1p and Stl2p are very different in size and do not show any appreciable homology. That Stl1p and Stl2p are functional homologues of Sec12p was confirmed by showing that expression of either cloned gene complements a sec12 null mutation. Our results indicate that some of the mechanisms regulating vesicle formation at the ER are conserved not only in yeasts, but also in plants.

Amino Acid Sequence↗
PubMed1992

Cloning of the mating-type gene MATA of the yeast Yarrowia lipolytica.

The mating type gene MATA of the dimorphic yeast Yarrowia lipolytica was cloned. The strategy used was based on the presumed function of this gene in the induction of sporulation. A diploid strain homozygous for the mating type B was transformed with an integrative gene bank from an A wild-type strain. A sporulating transformant was isolated, which contained a plasmid with an 11.6 kb insert. This sequence was rescued from the chromosomal DNA of the transformant and deletion mapping was performed to localize the MAT insert. The MAT gene conferred both sporulating and non-mating phenotypes on a B/B diploid. A LEU2 sequence targeted to this locus segregated like a mating type-linked gene. The A strain did not contain silent copies of the MAT gene.

Chromosome Deletion↗
PubMed1992

Hyperthermostable variants of a highly thermostable alpha-amylase.

Genetic screening at temperatures between 70-80 degrees C far exceeds the range of growth of most bacteria, and is not applicable to isolate easily thermostable protein variants. We describe a temperature shift protocol and an in vivo screening method which allowed us to identify a hyperthermostable variant of the thermostable alpha-amylase from Bacillus licheniformis. Our strategy was to select, after hydroxylamine mutagenesis, an intragenic suppressor mutation which overcomes a mutation leading to a thermolabile enzyme. Sequence analysis of the mutated gene revealed only one change in the amino acid sequence, substituting a valine for alanine at position 209. This single amino acid replacement increased the half-life of the protein at 90 degrees C by a factor of two to three relative to the wild-type enzyme. When this substitution was combined with another stabilizing substitution (H133Y) we described previously, the stabilizing effects were additive. The half-life of the new protein was about 12 hours at 90 degrees C, corresponding to a nine to ten-fold increase over the wild-type enzyme and the industrial Bacillus licheniformis alpha-amylase Termamyl. These mutations are located in a predicted folding domain of the protein which appears crucial in determining thermal stability.

Alanine↗
PubMed1991

Role of the proregion in the production and secretion of the Yarrowia lipolytica alkaline extracellular protease.

The yeast Yarrowia lipolytica secretes an alkaline extracellular protease (AEP). It is first synthesized as a precursor comprising a putative signal peptide, a stretch of 10 X-Ala or X-Pro sequences that are substrates for a dipeptidyl aminopeptidase, a large pro-region that contains a glycosylation site and two Lys-Arg sites that can be cleaved by a KEX2-like endoprotease and finally the mature protease itself. A defect in the XPR6 (KEX2-like) gene results in the secretion of an inactive proenzyme (Matoba, S., and Ogrydziak, D. M. (1989) J. Biol. Chem. 264, 6037-6043), showing that the proregion inhibits protease activity. To determine whether the proregion plays an additional role in protease secretion, we have generated deletions and point mutations in the corresponding region of the structural gene. In this paper we examine the effects of these mutations on AEP secretion and maturation and show that the proregion is essential for its secretion. All deletions affecting the proregion resulted in the intracellular accumulation of unprocessed precursors. Deletion of the glycosylation site in the proregion resulted in the production of an unglycosylated precursor that was secreted and matured correctly at 18 degrees C but accumulated in the cells at 28 degrees C. From these results, we propose that the AEP prosequence plays an additional essential role in guiding the proper folding of the protein into a conformation compatible with secretion.

Amino Acid Sequence↗