Publications by authors named "Carlos Gancedo"

21 Publications

  • Page 1 of 1

Construction and characterization of a Saccharomyces cerevisiae strain able to grow on glucosamine as sole carbon and nitrogen source.

Sci Rep 2018 11 16;8(1):16949. Epub 2018 Nov 16.

Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC-UAM. Calle Arturo Duperier 4, 28029, Madrid, Spain.

Saccharomyces cerevisiae can transport and phosphorylate glucosamine, but cannot grow on this amino sugar. While an enzyme catalyzing the reaction from glucosamine-6-phosphate to fructose-6-phosphate, necessary for glucosamine catabolism, is present in yeasts using N-acetylglucosamine as carbon source, a sequence homology search suggested that such an enzyme is absent from Saccharomyces cerevisiae. The gene YlNAG1 encoding glucosamine-6-phosphate deaminase from Yarrowia lipolytica was introduced into S. cerevisiae and growth in glucosamine tested. The constructed strain grew in glucosamine as only carbon and nitrogen source. Growth on the amino sugar required respiration and caused an important ammonium excretion. Strains overexpressing YlNAG1 and one of the S. cerevisiae glucose transporters HXT1, 2, 3, 4, 6 or 7 grew in glucosamine. The amino sugar caused catabolite repression of different enzymes to a lower extent than that produced by glucose. The availability of a strain of S. cerevisiae able to grow on glucosamine opens new possibilities to investigate or manipulate pathways related with glucosamine metabolism in a well-studied organism.
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http://dx.doi.org/10.1038/s41598-018-35045-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240059PMC
November 2018

55 years together-our life with yeasts.

FEMS Yeast Res 2017 11;17(7)

Instituto de Investigaciones Biomédicas 'Alberto Sols' CSIC-UAM, E-28029 Madrid, Spain.

The authors look back at their life together in yeast research, the influences that shaped it, certain challenges and changes in laboratory and funding policies.
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http://dx.doi.org/10.1093/femsyr/fox070DOI Listing
November 2017

The Expanding Landscape of Moonlighting Proteins in Yeasts.

Microbiol Mol Biol Rev 2016 09 27;80(3):765-77. Epub 2016 Jul 27.

Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid, Spain.

Moonlighting proteins are multifunctional proteins that participate in unrelated biological processes and that are not the result of gene fusion. A certain number of these proteins have been characterized in yeasts, and the easy genetic manipulation of these microorganisms has been useful for a thorough analysis of some cases of moonlighting. As the awareness of the moonlighting phenomenon has increased, a growing number of these proteins are being uncovered. In this review, we present a crop of newly identified moonlighting proteins from yeasts and discuss the experimental evidence that qualifies them to be classified as such. The variety of moonlighting functions encompassed by the proteins considered extends from control of transcription to DNA repair or binding to plasminogen. We also discuss several questions pertaining to the moonlighting condition in general. The cases presented show that yeasts are important organisms to be used as tools to understand different aspects of moonlighting proteins.
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http://dx.doi.org/10.1128/MMBR.00012-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981672PMC
September 2016

The gene YALI0E20207g from Yarrowia lipolytica encodes an N-acetylglucosamine kinase implicated in the regulated expression of the genes from the N-acetylglucosamine assimilatory pathway.

PLoS One 2015 27;10(3):e0122135. Epub 2015 Mar 27.

Department of Metabolism and Cell Signalling, Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC-UAM, Madrid, Spain.

The non-conventional yeast Yarrowia lipolytica possesses an ORF, YALI0E20207g, which encodes a protein with an amino acid sequence similar to hexokinases from different organisms. We have cloned that gene and determined several enzymatic properties of its encoded protein showing that it is an N-acetylglucosamine (NAGA) kinase. This conclusion was supported by the lack of growth in NAGA of a strain carrying a YALI0E20207g deletion. We named this gene YlNAG5. Expression of YlNAG5 as well as that of the genes encoding the enzymes of the NAGA catabolic pathway-identified by a BLAST search-was induced by this sugar. Deletion of YlNAG5 rendered that expression independent of the presence of NAGA in the medium and reintroduction of the gene restored the inducibility, indicating that YlNag5 participates in the transcriptional regulation of the NAGA assimilatory pathway genes. Expression of YlNAG5 was increased during sporulation and homozygous Ylnag5/Ylnag5 diploid strains sporulated very poorly as compared with a wild type isogenic control strain pointing to a participation of the protein in the process. Overexpression of YlNAG5 allowed growth in glucose of an Ylhxk1glk1 double mutant and produced, in a wild type background, aberrant morphologies in different media. Expression of the gene in a Saccharomyces cerevisiae hxk1 hxk2 glk1 triple mutant restored ability to grow in glucose.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122135PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4376941PMC
March 2016

The repressor Rgt1 and the cAMP-dependent protein kinases control the expression of the SUC2 gene in Saccharomyces cerevisiae.

Biochim Biophys Acta 2015 Jul 22;1850(7):1362-7. Epub 2015 Mar 22.

Department of Metabolism and Cell Signalling, Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC-UAM Arturo Duperier 4, E-28029 Madrid, Spain. Electronic address:

Background: A low level of glucose is required for maximal transcription of the SUC2 gene in Saccharomyces cerevisiae. Although the repressor Rgt1 binds the SUC2 promoter in gel-shift assays, it has been reported that Rgt1 has only minimal effects on SUC2 expression. Rgt1 acts together with Mth1 to repress the HXT genes encoding glucose transporters, and the release of Rgt1 from some HXT promoters requires cAMP-dependent protein kinase (PKA) activity.

Methods: The genes RGT1 and MTH1 have been disrupted and the SUC2 promoter modified in several S. cerevisiae backgrounds. Yeast cells were grown in different carbon sources in the presence or absence of 0.1 or 2% glucose, and invertase was assayed in whole cells.

Results: Galactose, glycerol or ethanol hindered invertase induction by low glucose, but lactate did not. During growth in lactate, deletion of RGT1 or MTH1 caused a marked increase in invertase levels, and elimination of the Rgt1-binding site in the SUC2 promoter caused also invertase induction. PKA activity decreased invertase levels in cells growing in lactate, and increased them during growth in lactate+0.1% glucose.

Conclusions: The low level of expression of SUC2 in the absence of glucose is mainly due to repression by the Rgt1-Mth1 complex. Repression is dependent on PKA activity, but not on any specific Tpk isoenzyme.

General Significance: The results show that previously overlooked regulatory elements, such as Rgt1 and Tpks, participate in the control of SUC2 expression in S.cerevisiae.
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http://dx.doi.org/10.1016/j.bbagen.2015.03.006DOI Listing
July 2015

Evolution of moonlighting proteins: insight from yeasts.

Biochem Soc Trans 2014 Dec;42(6):1715-9

*Department of Metabolism and Cell Signalling. Instituto de Investigaciones Biomédicas "Alberto Sols", CSIC-UAM, Madrid E-28029, Spain.

The present article addresses the possibilities offered by yeasts to study the problem of the evolution of moonlighting proteins. It focuses on data available on hexokinase from Saccharomyces cerevisiae that moonlights in catabolite repression and on galactokinase from Kluyveromyces lactis that moonlights controlling the induction of the GAL genes. Possible experimental approaches to studying the evolution of moonlighting hexose kinases are suggested.
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http://dx.doi.org/10.1042/BST20140199DOI Listing
December 2014

Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose 6-phosphate dynamics.

Biochem J 2013 Sep;454(2):227-37

Université de Toulouse, INSA, UPS, INP, France.

In Saccharomyces cerevisiae, synthesis of T6P (trehalose 6-phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H⁺-ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein from Yarrowia lipolytica, nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H⁺-ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism.
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http://dx.doi.org/10.1042/BJ20130587DOI Listing
September 2013

An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae.

Microb Cell Fact 2012 Sep 15;11:131. Epub 2012 Sep 15.

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

Background: Pyruvate-decarboxylase negative (Pdc⁻) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc⁻S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc⁻ strains cannot grow on high glucose concentrations and require C₂-compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry.

Results: Genetic analysis of a Pdc⁻ strain previously evolved to overcome these deficiencies revealed a 225 p in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc⁻ strain enabled growth on 20 g l⁻¹ glucose and 0.3% (v/v) ethanol at a maximum specific growth rate (0.24 h⁻¹) similar to that of the evolved Pdc⁻ strain (0.23 h⁻¹). Furthermore, the reverse engineered Pdc⁻ strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h⁻¹) than the evolved strain (0.20 h⁻¹). The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc⁻S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc⁻ strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C₂-compound auxotrophy.

Conclusions: In this study we have discovered and characterised a mutation in MTH1 enabling Pdc⁻ strains to grow on glucose as the sole carbon source. This successful example of reverse engineering not only increases the understanding of the glucose tolerance of evolved Pdc⁻ S. cerevisiae, but also allows introduction of this portable genetic element into various industrial yeast strains, thereby simplifying metabolic engineering strategies.
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http://dx.doi.org/10.1186/1475-2859-11-131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503853PMC
September 2012

Alberto Sols, teacher and mentor of spanish biochemists (1917-1989).

Authors:
Carlos Gancedo

IUBMB Life 2012 Jun 25;64(6):545-50. Epub 2012 Apr 25.

Instituto de Investigaciones, Biomédicas Alberto Sols CSIC-UAM., Department of Metabolism and Cell Signaling, 28029 Madrid, Spain.

Biochemistry in Spain owes much to the figure of Alberto Sols. In words of Nobel Prize winner Severo Ochoa: "He has been the first scientist to establish successfully biochemistry in Spain." His intellectual rigour, care in experimental design, emphasis on quality, and attention to the presentation of results permeated far beyond his inner circle to the then fledging Spanish biochemical community. It would be difficult to find some Spanish biochemist of the generation that now starts to retire who has not been influenced in a way or another by the work of Sols. However, it is also likely that the new generations of biochemists and molecular biologists in the country ignore who was Sols and what their field owns to him. The following lines try to highlight some key points of his scientific biography, the circumstances in which they took place and the state of the corresponding research area at that moment.
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http://dx.doi.org/10.1002/iub.1001DOI Listing
June 2012

Disruption of Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase does not affect growth in glucose but impairs growth at high temperature.

PLoS One 2011 12;6(9):e23695. Epub 2011 Sep 12.

Department of Metabolism and Cell Signalling, Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Madrid, Spain.

We have cloned the Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase by complementation of the lack of growth in glucose of a Saccharomyces cerevisiae tps1 mutant. Disruption of YlTPS1 could only be achieved with a cassette placed in the 3' half of its coding region due to the overlap of its sequence with the promoter of the essential gene YlTFC1. The Yltps1 mutant grew in glucose although the Y. lipolytica hexokinase is extremely sensitive to inhibition by trehalose-6-P. The presence of a glucokinase, insensitive to trehalose-6-P, that constitutes about 80% of the glucose phosphorylating capacity during growth in glucose may account for the growth phenotype. Trehalose content was below 1 nmol/mg dry weight in Y. lipolytica, but it increased in strains expressing YlTPS1 under the control of the YlTEF1 promoter or with a disruption of YALI0D15598 encoding a putative trehalase. mRNA levels of YlTPS1 were low and did not respond to thermal stresses, but that of YlTPS2 (YALI0D14476) and YlTPS3 (YALI0E31086) increased 4 and 6 times, repectively, by heat treatment. Disruption of YlTPS1 drastically slowed growth at 35°C. Homozygous Yltps1 diploids showed a decreased sporulation frequency that was ascribed to the low level of YALI0D20966 mRNA an homolog of the S. cerevisiae MCK1 which encodes a protein kinase that activates early meiotic gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023695PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3171402PMC
February 2012

Unraveling moonlighting functions with yeasts.

IUBMB Life 2011 Jul 13;63(7):457-62. Epub 2011 Apr 13.

Department of Metabolism and Cell Signaling, Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, Madrid, Spain.

This review considers the use of yeasts to study protein moonlighting functions. The cases discussed highlight the possibilities offered by the well-developed yeast genetics for the study of moonlighting mechanisms. The possibility to generate sets of mutants encoding different protein variants has allowed in some cases to map the regions that participate in the moonlighting function. We discuss cases of enzymes that moonlight in such different activities as control of transcription, assembly of multimeric proteins, stabilization of mitochondrial DNA or biosynthesis of CoA. The moonlighting role of an enzyme and its metabolic function seems to have evolved independently as indicated by the finding that a protein may moonlight in a yeast species but not in others. Yeasts may open ways to study possible evolutionary relationships among moonlighting proteins.
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http://dx.doi.org/10.1002/iub.454DOI Listing
July 2011

The gluconeogenic enzyme fructose-1,6-bisphosphatase is dispensable for growth of the yeast Yarrowia lipolytica in gluconeogenic substrates.

Eukaryot Cell 2008 Oct 8;7(10):1742-9. Epub 2008 Aug 8.

Department of Metabolism and Cell Signaling, Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Madrid, Spain

The genes encoding gluconeogenic enzymes in the nonconventional yeast Yarrowia lipolytica were found to be differentially regulated. The expression of Y. lipolytica FBP1 (YlFBP1) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of YlPCK1, encoding phosphoenolpyruvate carboxykinase, and YlICL1, encoding isocitrate lyase. We constructed Y. lipolytica strains with two different disrupted versions of YlFBP1 and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high K(m) (2.3 mM) for fructose-1,6-bisphosphate. The gene YlFBP1 restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a Saccharomyces cerevisiae fbp1 mutant, but the introduction of the FBP1 gene from S. cerevisiae in the Ylfbp1 mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus.
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http://dx.doi.org/10.1128/EC.00169-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2568072PMC
October 2008

Moonlighting proteins in yeasts.

Microbiol Mol Biol Rev 2008 Mar;72(1):197-210, table of contents

Department of Metabolism and Cell Signaling, Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM, 28029 Madrid, Spain.

Proteins able to participate in unrelated biological processes have been grouped under the generic name of moonlighting proteins. Work with different yeast species has uncovered a great number of moonlighting proteins and shown their importance for adequate functioning of the yeast cell. Moonlighting activities in yeasts include such diverse functions as control of gene expression, organelle assembly, and modification of the activity of metabolic pathways. In this review, we consider several well-studied moonlighting proteins in different yeast species, paying attention to the experimental approaches used to identify them and the evidence that supports their participation in the unexpected function. Usually, moonlighting activities have been uncovered unexpectedly, and up to now, no satisfactory way to predict moonlighting activities has been found. Among the well-characterized moonlighting proteins in yeasts, enzymes from the glycolytic pathway appear to be prominent. For some cases, it is shown that despite close phylogenetic relationships, moonlighting activities are not necessarily conserved among yeast species. Organisms may utilize moonlighting to add a new layer of regulation to conventional regulatory networks. The existence of this type of proteins in yeasts should be taken into account when designing mutant screens or in attempts to model or modify yeast metabolism.
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http://dx.doi.org/10.1128/MMBR.00036-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268286PMC
March 2008

How I became a biochemist.

Authors:
Carlos Gancedo

IUBMB Life 2006 Jan;58(1):59-61

Unidad de Bioquímica y Genética de Levaduras, Instituto de Investigaciones Biomédicas Alberto Sols, Madrid, Spain.

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http://dx.doi.org/10.1080/15216540500364107DOI Listing
January 2006

The dimorphic yeast Yarrowia lipolytica possesses an atypical phosphofructokinase: characterization of the enzyme and its encoding gene.

Microbiology (Reading) 2005 May;151(Pt 5):1465-1474

Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM and Departamento de Bioquímica, Facultad de Medicina de la Universidad Autónoma de Madrid, Arzobispo Morcillo 4, 28029 Madrid, Spain.

The phosphofructokinase from the non-conventional yeast Yarrowia lipolytica (YlPfk) was purified to homogeneity, and its encoding gene isolated. YlPfk is an octamer of 869 kDa composed of a single type of subunit, and shows atypical kinetic characteristics. It did not exhibit cooperative kinetics for fructose 6-phosphate (Hill coefficient, h 1.1; S0.5 52 microM), it was inhibited moderately by MgATP (Ki 3.5 mM), and it was strongly inhibited by phosphoenolpyruvate (Ki 61 microM). Fructose 2,6-bisphosphate did not activate the enzyme, and AMP and ADP were also without effect. The gene YlPFK1 has no introns, and encodes a putative protein of 953 aa, with a molecular mass consistent with the subunit size found after purification. Disruption of the gene abolished growth in glucose and Pfk activity, while reintroduction of the gene restored both properties. This indicates that Y. lipolytica has only one gene encoding Pfk, and supports the finding that the enzyme consists of identical subunits. Glucose did not interfere with growth of the Ylpfk1 disruptant in permissive carbon sources. The unusual kinetic characteristics of YlPfk, and the intracellular concentrations of glycolytic intermediates during growth in glucose, suggest that YlPfk may play an important role in the regulation of glucose metabolism in Y. lipolytica, different from the role played by the enzyme in Saccharomyces cerevisiae.
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http://dx.doi.org/10.1099/mic.0.27856-0DOI Listing
May 2005

Yarrowia lipolytica mutants devoid of pyruvate carboxylase activity show an unusual growth phenotype.

Eukaryot Cell 2005 Feb;4(2):356-64

Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-Universidad Autónoma de Madrid, Madrid, Spain.

We have cloned and characterized the gene PYC1, encoding the unique pyruvate carboxylase in the dimorphic yeast Yarrowia lipolytica. The protein putatively encoded by the cDNA has a length of 1,192 amino acids and shows around 70% identity with pyruvate carboxylases from other organisms. The corresponding genomic DNA possesses an intron of 269 bp located 133 bp downstream of the starting ATG. In the branch motif of the intron, the sequence CCCTAAC, not previously found at this place in spliceosomal introns of Y. lipolytica, was uncovered. Disruption of the PYC1 gene from Y. lipolytica did not abolish growth in glucose-ammonium medium, as is the case in other eukaryotic microorganisms. This unusual growth phenotype was due to an incomplete glucose repression of the function of the glyoxylate cycle, as shown by the lack of growth in that medium of double pyc1 icl1 mutants lacking both pyruvate carboxylase and isocitrate lyase activity. These mutants grew when glutamate, aspartate, or Casamino Acids were added to the glucose-ammonium medium. The cDNA from the Y. lipolytica PYC1 gene complemented the growth defect of a Saccharomyces cerevisiae pyc1 pyc2 mutant, but introduction of either the S. cerevisiae PYC1 or PYC2 gene into Y. lipolytica did not result in detectable pyruvate carboxylase activity or in growth on glucose-ammonium of a Y. lipolytica pyc1 icl1 double mutant.
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http://dx.doi.org/10.1128/EC.4.2.356-364.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC549329PMC
February 2005

The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi.

FEMS Yeast Res 2004 Jan;4(4-5):351-9

Albert Sols Institute of Biomedical Research, CSIC-UAM, C/ Arturo Duperier 4, 28029 Madrid, Spain.

The view of the role of trehalose in yeast has changed in the last few years. For a long time considered a reserve carbohydrate, it gained new importance when its function in the acquisition of thermotolerance was demonstrated. More recently the cellular processes in which the trehalose biosynthetic pathway has been implicated range from the control of glycolysis to sporulation and infectivity by certain fungal pathogens. There is now enough experimental evidence to conclude that trehalose 6-phosphate, an intermediate of trehalose biosynthesis, is an important metabolic regulator in such different organisms as yeasts or plants. Its inhibition of hexokinase plays a key role in the control of the glycolytic flux in Saccharomyces cerevisiae but other, likely important, sites of action are still unknown. We present examples of the phenotypes produced by mutations in the two steps of the trehalose biosynthetic pathway in different yeasts and fungi, and whenever possible examine the molecular explanations advanced to interpret them.
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http://dx.doi.org/10.1016/S1567-1356(03)00222-8DOI Listing
January 2004

Mitochondrial localization of the mevalonate pathway enzyme 3-Hydroxy-3-methyl-glutaryl-CoA reductase in the Trypanosomatidae.

Mol Biol Cell 2004 Mar 29;15(3):1356-63. Epub 2003 Dec 29.

Instituto de Parasitología y Biomedicina "López-Neyra", Consejo Superior de Investigaciones Científicas, 18001 Granada, Spain.

3-Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is a key enzyme in the sterol biosynthesis pathway, but its subcellular distribution in the Trypanosomatidae family is somewhat controversial. Trypanosoma cruzi and Leishmania HMGRs are closely related in their catalytic domains to bacterial and eukaryotic enzymes described but lack an amino-terminal domain responsible for the attachment to the endoplasmic reticulum. In the present study, digitonin-titration experiments together with immunoelectron microscopy were used to establish the intracellular localization of HMGR in these pathogens. Results obtained with wild-type cells and transfectants overexpressing the enzyme established that HMGR in both T. cruzi and Leishmania major is localized primarily in the mitochondrion and that elimination of the mitochondrial targeting sequence in Leishmania leads to protein accumulation in the cytosolic compartment. Furthermore, T. cruzi HMGR is efficiently targeted to the mitochondrion in yeast cells. Thus, when the gene encoding T. cruzi HMGR was expressed in a hmg1 hmg2 mutant of Saccharomyces cerevisiae, the mevalonate auxotrophy of mutant cells was relieved, and immunoelectron analysis showed that the parasite enzyme exhibits a mitochondrial localization, suggesting a conservation between the targeting signals of both organisms.
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http://dx.doi.org/10.1091/mbc.e03-10-0720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC363142PMC
March 2004

New mutations of Saccharomyces cerevisiae that partially relieve both glucose and galactose repression activate the protein kinase Snf1.

FEMS Yeast Res 2003 Mar;3(1):77-84

Instituto de Investigaciones Biomédicas 'Alberto Sols' CSIC-UAM, Unidad de Bioquímica y Genética de Levaduras, C/Arturo Duperier 4, 28029, Madrid, Spain.

We isolated from Saccharomyces cerevisiae two mutants, esc1-1 and ESC3-1, in which genes FBP1, ICL1 or GDH2 were partially derepressed during growth in glucose or galactose. The isolation was done starting with a triple mutant pyc1 pyc2 mth1 unable to grow in glucose-ammonium medium and selecting for mutants able to grow in the non-permissive medium. HXT1 and HXT2 which encode glucose transporters were expressed at high glucose concentrations in both esc1-1 and ESC3-1 mutants, while derepression of invertase at low glucose concentrations was impaired. REG1, cloned as a suppressor of ESC3-1, was not allelic to ESC3-1. Two-hybrid analysis showed an increased interaction of the protein kinase Snf1 with Snf4 in the ESC3-1 mutant; this was not due to mutations in SNF1 or SNF4. ESC3-1 did not bypass the requirement of Snf1 for derepression. We hypothesize that ESC3-1 either facilitates activation of Snf1 or interferes with its glucose-dependent inactivation.
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http://dx.doi.org/10.1111/j.1567-1364.2003.tb00141.xDOI Listing
March 2003

Pyruvate carboxylase is an essential protein in the assembly of yeast peroxisomal oligomeric alcohol oxidase.

Mol Biol Cell 2003 Feb;14(2):786-97

Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands.

Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.
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http://dx.doi.org/10.1091/mbc.e02-07-0417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150008PMC
February 2003

Disruption in Candida albicans of the TPS2 gene encoding trehalose-6-phosphate phosphatase affects cell integrity and decreases infectivity.

Microbiology (Reading) 2002 May;148(Pt 5):1281-90

Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM, Unidad de Bioquímica y Genética de Levaduras, 28029 Madrid, Spain.

The gene CaTPS2 encoding trehalose-6-phosphate (T6P) phosphatase from Candida albicans has been cloned and disrupted in this organism. The Catps2/Catps2 mutant did not accumulate trehalose but accumulated high levels of T6P. Disruption of the two copies of the CaTPS2 gene did not abolish growth even at 42 degrees C, but decreased the growth rate. In the stationary phase, the Catps2/Catps2 mutant aggregated, more than 50% of its cells became permeable to propidium iodide and a large amount of protein was found in the culture medium. Aggregation occurred only at pH values higher than 7 and was avoided by osmoprotectants; it was never observed during the exponential phase of growth. The mutant formed colonies with a smooth border on Spider medium. Mice inoculated with 1.5 x 10(6) c.f.u. of wild-type cells died after 8 days, while 80% of those inoculated with the same number of c.f.u. of the Catps2/Catps2 mutant survived for at least 1 month. Reintroduction of the wild-type CaTPS2 gene in the Catps2/Catps2 mutant abolished the phenotypes described. It is hypothesized that the accumulation of T6P interferes with the assembly of a normal cell wall.
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http://dx.doi.org/10.1099/00221287-148-5-1281DOI Listing
May 2002