Abstract
Many transcription factors contribute to cellular homeostasis by integrating multiple signals. Signaling via the yeast Gal80 protein, a negative regulator of the prototypic transcription activator Gal4, is primarily regulated by galactose. ScGal80 from Saccharomyces cerevisiae has been reported to bind NAD(P). Here, we show that the ability to bind these ligands is conserved in KlGal80, a Gal80 homolog from the distantly related yeast Kluyveromyces lactis. However, the homologs apparently have diverged with respect to response to the dinucleotide. Strikingly, ScGal80 binds NAD(P) and NAD(P)H with more than 50-fold higher affinity than KlGal80. In contrast to ScGal80, where NAD is neutral, NAD and NADP have a negative effect in KlGal80 on its interaction with a KlGal4-peptide in vitro. Swapping a loop in the NAD(P) binding Rossmann-fold of ScGal80 into KlGal80 increases the affinity for NAD(P) and has a significant impact on KlGal4 regulation in vivo. Apparently, dinucleotide binding allows coupling of the metabolic state of the cell to regulation of the GAL/LAC genes. The particular sequences involved in binding determine how exactly the metabolic state is sensed and integrated by Gal80 to regulate Gal4.
Acknowledgments
We thank Dr. Renate Langhammer and Ursula Klokow for help in strain constructions and are grateful to all our colleagues from the DFG Doctoral Reasearch Training Group 1026 for many helpful discussions. Technical assistance by Karin Sorge is gratefully acknowledged. We are grateful to Victor Sourjik (University of Heidelberg) for being able to use the microscope.This work was supported by DFG Graduiertenkolleg GRK 1026 (Project B.1) und DFG grant BR921/4 to K.D.B.
References
Agashe, V.R., Schmid, F.X., and Udgaonkar, J.B. (1997). Thermodynamics of the complex protein unfolding reaction of barstar. Biochemistry 36, 12288–12295.10.1021/bi971062dSearch in Google Scholar
Anders, A., Lilie, H., Franke, K., Kapp, L., Stelling, J., Gilles, E.D., and Breunig, K.D. (2006). The galactose switch in Kluyveromyces lactis depends on nuclear competition between Gal4 and Gal1 for Gal80 binding. J. Biol. Chem. 281, 29337–29348.10.1074/jbc.M604271200Search in Google Scholar
Aravind, L. and Koonin, E.V. (1998). Eukaryotic transcription regulators derive from ancient enzymatic domains. Curr. Biol. 8, R111–R113.10.1016/S0960-9822(98)70982-0Search in Google Scholar
Belenky, P., Racette, F.G., Bogan, K.L., McClure, J.M., Smith, J.S., and Brenner, C. (2007). Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+. Cell 129, 473–484.10.1016/j.cell.2007.03.024Search in Google Scholar
Belenky, P.A., Moga, T.G., and Brenner, C. (2008). Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1. J. Biol. Chem. 283, 8075–8079.10.1074/jbc.C800021200Search in Google Scholar
Breunig, K.D. and Kuger, P. (1987). Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site. Mol. Cell. Biol. 7, 4400–4406.Search in Google Scholar
Carpenter, A.E., Jones, T.R., Lamprecht, M.R., Clarke, C., Kang, I.H., Friman, O., Guertin, D.A., Chang, J.H., Lindquist, R.A., Moffat, J., Golland, P., and Sabatini, D.M. (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biology 7:R100. Cell 127, 579–589.Search in Google Scholar
Cho, Y.H., Yoo, S.D., and Sheen, J. (2006). Regulatory functions of nuclear hexokinase1 complex in glucose signaling. Cell 127, 579–589.10.1016/j.cell.2006.09.028Search in Google Scholar
Dickson, R.C., Gerardot, C.J., and Martin, A.K. (1990). Genetic evidence for similar negative regulatory domains in the yeast transcription activators GAL4 and LAC9. Nucleic Acids Res. 18, 5213–5217.10.1093/nar/18.17.5213Search in Google Scholar
Eftink, M.R. and Ghiron, C.A. (1981). Fluorescence quenching studies with proteins. Anal. Biochem. 114, 199–227.10.1016/0003-2697(81)90474-7Search in Google Scholar
Hittinger, C.T. and Carroll, S.B. (2007). Gene duplication and the adaptive evolution of a classic genetic switch. Nature 449, 677–681.10.1038/nature06151Search in Google Scholar PubMed
Hittinger, C.T., Rokas, A., and Carroll, S.B. (2004). Parallel inactivation of multiple GAL pathway genes and ecological diversification in yeasts. Proc. Natl. Acad. Sci. USA 101, 14144–14149.10.1073/pnas.0404319101Search in Google Scholar PubMed PubMed Central
Houser, J.R., Ford, E., Chatterjea, S.M., Maleri, S., Elston, T.C., and Errede, B. (2012). An improved short-lived fluorescent protein transcriptional reporter for Saccharomyces cerevisiae. Yeast 29, 519–530.10.1002/yea.2932Search in Google Scholar
Imai, S., Armstrong, C.M., Kaeberlein, M., and Guarente, L. (2000). Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795–800.10.1038/35001622Search in Google Scholar
Johnston, S.A., Salmeron, J.M., Jr., and Dincher, S.S. (1987). Interaction of positive and negative regulatory proteins in the galactose regulon of yeast. Cell 50, 143–146.10.1016/0092-8674(87)90671-4Search in Google Scholar
Keogh, R.S., Seoighe, C., and Wolfe, K.H. (1998). Evolution of gene order and chromosome number in Saccharomyces, Kluyveromyces and related fungi. Yeast 14, 443–457.10.1002/(SICI)1097-0061(19980330)14:5<443::AID-YEA243>3.0.CO;2-LSearch in Google Scholar
Kim, J.W. and Dang, C.V. (2005). Multifaceted roles of glycolytic enzymes. Trends Biochem. Sci. 30, 142–150.10.1016/j.tibs.2005.01.005Search in Google Scholar
Kingston, R.L., Scopes, R.K., and Baker, E.N. (1996). The structure of glucose-fructose oxidoreductase from Zymomonas mobilis: an osmoprotective periplasmic enzyme containing non-dissociable NADP. Structure. 4, 1413–1428.10.1016/S0969-2126(96)00149-9Search in Google Scholar
Krijger, J.J., Baumann, J., Wagner, M., Schulze, K., Reinsch, C., Klose, T., Onuma, O.F., Simon, C., Behrens, S.E., and Breunig, K.D. (2012). A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in kluyveromyces lactis. Microb. Cell Fact. 11, 112.10.1186/1475-2859-11-112Search in Google Scholar
Kumar, P.R., Yu, Y., Sternglanz, R., Johnston, S.A., and Joshua-Tor, L. (2008). NADP regulates the yeast GAL induction system. Science 319, 1090–1092.10.1126/science.1151903Search in Google Scholar
Kurtzman, C.P. (2003). Phylogenetic circumscription of Saccharomyces, Kluyveromyces and other members of the Saccharomycetaceae, and the proposal of the new genera Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora. FEMS Yeast Res. 4, 233–245.10.1016/S1567-1356(03)00175-2Search in Google Scholar
Lamb, H.K., Leslie, K., Dodds, A.L., Nutley, M., Cooper, A., Johnson, C., Thompson, P., Stammers, D.K., and Hawkins, A.R. (2003). The negative transcriptional regulator NmrA discriminates between oxidized and reduced dinucleotides. J. Biol. Chem. 278, 32107–32114.10.1074/jbc.M304104200Search in Google Scholar
Lavy, T., Kumar, P.R., He, H., and Joshua-Tor, L. (2012). The Gal3p transducer of the GAL regulon interacts with the Gal80p repressor in its ligand-induced closed conformation. Genes Dev. 26, 294–303.10.1101/gad.182691.111Search in Google Scholar
Lehrer, S.S. (1971). Solute perturbation of protein fluorescence. The quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion. Biochemistry 10, 3254–3263.10.1021/bi00793a015Search in Google Scholar
Li, Y.F. and Bao, W.G. (2007). Why do some yeast species require niacin for growth? Different modes of NAD synthesis. FEMS Yeast Res. 7, 657–664.10.1111/j.1567-1364.2007.00231.xSearch in Google Scholar
Li, Y., Chen, G., and Liu, W. (2010). Alterations in the interaction between GAL4 and GAL80 effect regulation of the yeast GAL regulon mediated by the F box protein Dsg1. Curr. Microbiol. 61, 210–216.10.1007/s00284-010-9598-1Search in Google Scholar
Lin, S.J. and Guarente, L. (2003). Nicotinamide adenine dinucleotide, a metabolic regulator of transcription, longevity and disease. Curr. Opin. Cell Biol. 15, 241–246.10.1016/S0955-0674(03)00006-1Search in Google Scholar
Liou, G.G., Tanny, J.C., Kruger, R.G., Walz, T., and Moazed, D. (2005). Assembly of the SIR complex and its regulation by O-acetyl-ADP-ribose, a product of NAD-dependent histone deacetylation. Cell 121, 515–527.10.1016/j.cell.2005.03.035Search in Google Scholar
Melcher, K. (2005). Mutational hypersensitivity of a gene regulatory protein: Saccharomyces cerevisiae Gal80p. Genetics 171, 469–476.10.1534/genetics.105.045237Search in Google Scholar
Messenguy, F. and Dubois, E. (2003). Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development. Gene 316, 1–21.10.1016/S0378-1119(03)00747-9Search in Google Scholar
Meyer, J., Walker-Jonah, A., and Hollenberg, C.P. (1991). Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae. Mol. Cell. Biol. 11, 5454–5461.Search in Google Scholar
Moreno, F., Ahuatzi, D., Riera, A., Palomino, C.A., and Herrero, P. (2005). Glucose sensing through the Hxk2-dependent signalling pathway. Biochem. Soc. Trans. 33, 265–268.10.1042/BST0330265Search in Google Scholar PubMed
R Core Team (2013). R: A Language and Environment for Statistical Computing. (Vienna, Austria: R Foundation for Statistical Computing).Search in Google Scholar
Reece, R.J. and Platt, A. (1997). Signalling activation and repression of RNA polymerase II transcription in yeast. BioEssays 19, 1001–1010.10.1002/bies.950191110Search in Google Scholar PubMed
Riley, M.I., Hopper, J.E., Johnston, S.A., and Dickson, R.C. (1987). GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon. Mol. Cell. Biol. 7, 780–786.Search in Google Scholar
Rolland, F., Winderickx, J., and Thevelein, J.M. (2001). Glucose-sensing mechanisms in eukaryotic cells. Trends Biochem. Sci. 26, 310–317.10.1016/S0968-0004(01)01805-9Search in Google Scholar
Salmeron, J.M. and Johnston, S.A. (1986). Analysis of the Kluyveromyces lactis positive regulatory gene LAC9 reveals functional homology to, but sequence divergence from, the Saccharomyces cerevisiae GAL4 gene. Nucleic Acids Res. 14, 7767–7781.10.1093/nar/14.19.7767Search in Google Scholar
Salmeron, J.M., Langdon, S.D., and Johnston, S.A. (1989). Interaction between transcriptional activator protein LAC9 and negative regulatory protein GAL80. Mol. Cell. Biol. 9, 2950–2956.Search in Google Scholar
Schaffrath, R. and Breunig, K.D. (2000). Genetics and molecular physiology of the yeast Kluyveromyces lactis. Fungal. Genet. Biol. 30, 173–190.10.1006/fgbi.2000.1221Search in Google Scholar
Schmidt, D. (2010). Biophysikalische und molekulargenetische Untersuchung der Dinukleotid-Bindung von Gal80 aus Saccharomyces cerevisiae und Kluyveromyces lactis. Dissertation, Martin-Luther University Halle-Wittenberg.Search in Google Scholar
Sil, A.K., Alam, S., Xin, P., Ma, L., Morgan, M., Lebo, C.M., Woods, M.P., and Hopper, J.E. (1999). The Gal3p-Gal80p-Gal4p transcription switch of yeast: Gal3p destabilizes the Gal80p-Gal4p complex in response to galactose and ATP. Mol. Cell. Biol. 19, 7828–7840.10.1128/MCB.19.11.7828Search in Google Scholar
Thoden, J.B., Sellick, C.A., Timson, D.J., Reece, R.J., and Holden, H.M. (2005). Molecular structure of Saccharomyces cerevisiae Gal1p, a bifunctional galactokinase and transcriptional inducer. J. Biol. Chem. 280, 36905–36911.10.1074/jbc.M508446200Search in Google Scholar
Thoden, J.B., Sellick, C.A., Reece, R.J., and Holden, H.M. (2007). Understanding a transcriptional paradigm at the molecular level: the structure of yeast Gal80p. J. Biol. Chem. 282, 1534–1538.10.1074/jbc.C600285200Search in Google Scholar
Thoden, J.B., Ryan, L.A., Reece, R.J., and Holden, H.M. (2008). The interaction between an acidic transcriptional activator and its inhibitor. The molecular basis of Gal4p recognition by Gal80p. J. Biol. Chem. 283, 30266–30272.10.1074/jbc.M805200200Search in Google Scholar
Vaziri, H., Dessain, S.K., Ng Eaton, E., Imai, S.I., Frye, R.A., Pandita, T.K., Guarente, L., and Weinberg, R.A. (2001). hSIR2(SIRT1). functions as an NAD-dependent p53 deacetylase. Cell 107, 149–159.10.1016/S0092-8674(01)00527-XSearch in Google Scholar
Wickham, H. (2009). ggplot2: elegant graphics for data analysis, (New York: Springer).10.1007/978-0-387-98141-3Search in Google Scholar
Yano, K. and Fukasawa, T. (1997). Galactose-dependent reversible interaction of Gal3p with Gal80p in the induction pathway of Gal4p-activated genes of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 94, 1721–1726.10.1073/pnas.94.5.1721Search in Google Scholar PubMed PubMed Central
Zenke, F., Zachariae, W., Lunkes, A., and Breunig, K.D. (1993). Gal80 proteins of Kluyveromyces lactis and Saccharomyces cervisiae are highly conserved but contribute differently to glucose repression of the galactose regulon. Mol. Cell. Biol. 13, 7566–7576.Search in Google Scholar
Zenke, F.T., Engels, R., Vollenbroich, V., Meyer, J., Hollenberg, C.P., and Breunig, K.D. (1996). Activation of Gal4p by galactose-dependent interaction of galactokinase and Gal80p. Science 272, 1662–1665.10.1126/science.272.5268.1662Search in Google Scholar
Zenke, F.T., Kapp, L., and Breunig, K.D. (1999). Regulated phosphorylation of the Gal4p inhibitor Gal80p of Kluyveromyces lactis revealed by mutational analysis. Biol. Chemistry 380, 419–430.10.1515/BC.1999.056Search in Google Scholar
Zhang, Q., Piston, D.W., and Goodman, R.H. (2002). Regulation of corepressor function by nuclear NADH. Science 295, 1895–1897.10.1126/science.1069300Search in Google Scholar
Zheng, L., Roeder, R.G., and Luo, Y. (2003). S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 114, 255–266.10.1016/S0092-8674(03)00552-XSearch in Google Scholar
Supplemental Material: The online version of this article (DOI 10.1515/hsz-2014-0152) offers supplementary material, available to authorized users.
©2014 by Walter de Gruyter Berlin/Boston
