Publications by authors named "Carmen-Lisset Flores"

16 Publications

  • Page 1 of 1

Interaction between KDELR2 and HSP47 as a Key Determinant in Osteogenesis Imperfecta Caused by Bi-allelic Variants in KDELR2.

Am J Hum Genet 2020 11 13;107(5):989-999. Epub 2020 Oct 13.

Department of Clinical Genetics, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam 1081BT, the Netherlands.

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.
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http://dx.doi.org/10.1016/j.ajhg.2020.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675035PMC
November 2020

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

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

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

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

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

Sampling Saccharomyces cerevisiae cells by rapid filtration improves the yield of mRNAs.

FEMS Yeast Res 2004 May;4(7):751-6

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

To optimize the recovery of mRNAs extracted from yeast, different methods for sampling the yeast cells have been compared. For Saccharomyces cerevisiae strains growing on gluconeogenic carbon sources (derepressed cells) rapid filtration allowed much higher yields (3-10 fold) than centrifugation at room temperature or at 4 degrees C. Recovery of total RNA was similar with the different procedures. For S. cerevisiae growing on glucose, filtration caused a 2-4 fold improvement on the mRNA yields obtained from cells sampled by centrifugation. It was also observed that, when derepressed cells of S. cerevisiae W303-1A were collected by filtration and flash-frozen, part of the 25S and 18S rRNAs (up to 50%) was recovered in an unprocessed 32S or 33S form.
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http://dx.doi.org/10.1016/j.femsyr.2004.02.004DOI Listing
May 2004

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