Poster apresentado em 2004 Yeast Genetics and Molecular Biology Meeting, Seattle, USA, 27 Jul. - 1 Ag. 2004Portuguese Government - Ph.D. Grant from the (BD/1898/98)FEBS - short term fellowshipPortuguese Government - Technichal Grant POCTI/BME/36625/2000 , POCTI/1999/3662

Barbosa, Sónia

Casal, Margarida

Devaux, Frederic

Jacq, Claude

Paiva, Sandra

Universidade do Minho: RepositoriUM

Ady2p is essencial for the acetate permease activity in the yeastSaccharomyces cerevisiaeSandra Paiva*, Frederic Devaux*, Sónia Barbosa, Claude Jacq and Margarida Casal Centro/Departamento de Biologia, Universidade do Minho, 4710-057 Braga, PortugalLaboratoire de Génétique Moléculaire, CNRS UMR8541, Ecole Normale Supérieure, 46 Rue d’ Ulm, 75230 Paris Cedex 05, France*the authors contributted equally to the workSeveral yeast species, including Saccharomyces cerevisiae, are able to utilize acetic acid as a sole carbon and energy source under aerobic conditions (Barnett et al., 1990), acetate being a normal and frequent end-product of fermentation (Flores et al., 2000). When cells of S. cerevisiae are grown on respiratory substrates, activity for at least two monocarboxylate proton symporters are found, with differences in their mechanisms of regulation and specificity (Casal et al., 1996). A lactate–pyruvate-acetate-propionate transporter, induced in lactic or pyruvic acid-grown cells, is encoded by the gene JEN1 (Casal et al., 1999). In addition to Jen1p, another permease, which accepts acetate, propionate or formate, is present in cells grown in non-fermentable carbon sources (Casal et al., 1996; Makuc et al., 2001). To identify new genes involved in acetate uptake in S. cerevisiae, an analysis of the gene expression profiles of cells shifted from glucose to acetic acid was performed. This strategy allowed us to identify the membrane protein Ady2p as an essential component of the acetate active transport in these conditions and a valuable candidate as a new acetate transporter in yeast.The transcriptional adaptation of yeast cells to a shift from glucose to non-toxic conditions of acetic acid was characterized. These conditions of metabolic shift revealed to be very powerful for functional assignment of unknown proteins and for the study of mechanisms like translation regulation or mitochondria biogenesis.¾ADY2 gene is an essential component of acetate transport system  in S. cerevisiaeBarnett, JA. et al., 1990. Yeast. University Press; Casal, M. et al., 1996. Microbiology, 142: 1385-1390; Casal, M. et al., 1999. J.  Bacteriol., 181: 2620-2623; Flores, CL. et al., 2000. FEMS Microbiol. Rev., 24: 507-529; Makuc, J. et al., 2001. Yeast, 18: 1131-1143.Sandra Paiva received a Ph.D. Grant from the Portuguese Government (BD/1898/98) and received a FEBS short term fellowship for the work carried out in Paris. Sónia Barbosa received a Technichal Grant from a project (POCTI/BME/36625/2000) financed by the Portuguese Government. This work was suported by the project POCTI/1999/36625.Comparison of gene expressionprofiles between W303-1A and ady2Δ strainsAfter 4 hours in acetate, the ady2Δ strain does not have a dramatic difference in the global transcriptional response compared to the wild type suggesting that ADY2 is not a key regulator of the transcriptional response to acetic acid Adaptation of S. cerevisiae W303-1A and ady2Δ strains to acetic acid (0,5%, pH 6.0)Cells were cultivated in glucose and transferred to a medium containing acetic acid(0,5%, pH 6.0)¾ Cells of the ady2Δ have a normal growth in acetic acid whencompared with the wild type strain- W303-1A (ADY2)- W303-1A (ady2Δ)¾ ADY2-deleted strain (■) exhibited a dramatic decrease in the activity ofthe acetate premease when compared to the wt strain (S)¾ In the ADY2-deleted strain acetic acid crossed the plasma membraneby a simple diffusion mechanismADY2/YCR010c is an essencial gene for the mediatedtransport of acetic acid in S. cerevisiaeInitial uptake rates of labelled acetic acid pH 5.0 by 6 h of acetic acid-derepressed cellsW303-1A (ADY2)W303-1A (ady2Δ)Acetate is transported by a mediated transport system¾The activity of the acetate permease was present only after 6 h of induction¾The deletion of JEN1 did not affect the ability of the cells to transport acetic acid by a mediated mechanismExpression changes in genes encodingknown or putative transporters after 4h inacetic acid¾Genes YCR010c/ADY2, YNR002c/FUN34 and YDR384c/ATO3, homologous to the GPR1 gene of Yarrowia lipolytica were clearly activatedAnalysis of acetate uptake in S. cerevisiae ady2Δ containingthe gene ADY2 in the plasmid p416GPD0.0 0.5 1.0 1.5 2.00126 h4 h[acetic acid]V(nmol/ s / mg d. w.)YNBgluc YNBace4h           6H0.48 0.721.5 2.4Km(mM)Vmax(nmols-1mgdw-1)Apa IEco RVPst IKpn IMCSSac IURA 3CYC1 - TerminatorGPD PromotorAmpCEN6/ARSH4p416 GPD5804 bpPresence of a mediated transport system for acetic acidExpression of ADY2 in different carbon sourcesPyruvicac. 4hFormic ac. 4hGlucoseLactic ac. 4hAceticac. 4hEthanol 4hFormic ac. 6hPropionicac. 4hPropionicac. 6hGlycerolADY2JEN 1PDA 1Northern-blot analysis of  JEN1 and ADY2 transcripts from W303.1A strain. Total cellular RNA was prepared from cells growing exponentially in YNB media containing different carbon sourcesGlucose-grown cells Shift to acetic acid medium, 4 hGlucose 6PFructose 6PFructose 1,6PGlyceraldehyde 3PGlycerate 1,3PGlycerate 3PGlycerate 2PPEPPyruvateGlycerone PGlucoseTréhaloseAcetyl CoAOxalosuccinateOxaloacetateCitrateIsocitrateOxoglutarateSuccinyl CoASuccinate FumarateMalateHXK1,2GLK1FBP1PYC1PCK1CarnitineCAT2CRC1YAT1,2ethanolAcetaldehydeLactateDLD1PropanoatePDH1ACS1ACO1CIT3ICL2ADH2PDC1,5,6PGI1PFK1FBA1TPI1TDH1,2,3PGK1GPM1ENO1,2PYK2TPS3GLYCOLYSISIDP3IDH1,2KGD1,2LSC2 FUM1IDP3Complex IISDH 1,2,3YJL045WTCA CYCLECIT1,2,3ACO1MDH1,2GlyoxylateICL1,2MLS1DAL7GLYOXYLATECYCLEComplex INDI1YMR145CYDL085WComplex IIICOR1, QCR8, QCR7,RIP1, QCR2, QCR6, CYT1Complex IVCOX4, COX5A, COX6,COX8, COX9, COX12,COX13, COX17Complex VATP1, ATP2, ATP3, ATP4, ATP5, ATP7, ATP14, ATP15, ATP16, ATP17, ATP20OXIDATIVE PHOSPHORYLATIONHèmeHEM2, HEM3, HEM12COX11, COX10CYT2, FMC1, CYC3Acetyl CoAACH1ACS1Fatty AcidsPOX1 PEX11 CTA1 ECI1 FOX2 POT1 FAA3 OLE1 ACP1 IDP3ADR1YGR067c+HAP4, HAP3++ CAT8, SIP4++SIP2SNF1+AcetateROX1- GLC7-After 4 h induction { W303-1A (JEN1)zW303-1A (jen1Δ)After 6 h inductionUW303-1A (JEN1) W303-1A (jen1Δ)Setting up the best conditions for microarray analysesGlucose-grown cells Shift to YNB-acetic acid medium, pH 6.0

Ady2p is essential for the acetate pemease activity in the yeast Saccharomyces cerevisae

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Ady2p is essential for the acetate pemease activity in the yeast Saccharomyces cerevisae

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