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Yeast as a model to study eukaryotic transport systems

Yeast as a model to study eukaryotic transport systems. Hana Sychrová. Department of Membrane Transport Institute of Physiology, AS CR, v.v.i. Videnska 1083, 142 20 Prague 4 Tel: (420) 241 062 667 E-mail: sychrova @ biomed.cas.cz. Characterization of transport systems.

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Yeast as a model to study eukaryotic transport systems

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  1. Yeast as a model to study eukaryotic transport systems Hana Sychrová Department of Membrane Transport Institute of Physiology, AS CR, v.v.i. Videnska 1083, 142 20 Prague 4 Tel: (420) 241 062 667 E-mail: sychrova@biomed.cas.cz

  2. Characterization of transport systems • Kinetic parameters - KT , Jmax , ( sin/sout ), substrate specificity • pH optimum, inhibitors, source of energy (active transport) • Isolation of the geneand sequencingprimary structure and properties of the protein • Mutagenesis to identify crucial aa residues, secondary and tertiary structures • Protein purification reconstitution, crystalization • existence of similar systems (overlap of substrate specificity) • small amount of protein (low expression, short half-life) • denaturation of protein during membrane solubilization = loss of characteristic properties  expression of the gene in host organisms lacking their own transporters (yeast Saccharomyces cerevisiae)

  3. Saccharomyces cerevisiae • eukaryotic cell, 4 - 8 m, gen. time 2 h • haploid/diploid (2 x 16 chr, sequenced 1996) •  6 000 genes • simple isolation of mutants, viable with multiple mutations • universal transformation systems, easy disruption and deletion of genes chromosomal structure regulation of cell cycle and gene expressionbiogenesis and degradation of proteins (secretory pathway) protein structure/function relationshipinteraction between proteins (two hybrid screening system)cloning of long heterologous DNA fragments (YeastArtificialChromosome)expression of heterologous proteins Heterologous expression of transporters: characterization of own systems gene deletions  „non-transporting mutants“ expression and characterization of transporters from closely related organisms other organisms

  4. Na+/H+-antiporters All organisms Na+ out / H+ inbacteria (EcNhaA crystalized) yeast (3,PM and organelles) plants (>20, 2 subfamilies, PM and organelles) Elimination of surplus cations from cytosolu, Source of energy – H+ gradient across PM H+ out / Na+inanimal cells (NHE1-9, PM and organellles) Regulation of intracellular pH (elimination of surplus H+ from cytosol), Source of energy – Na+ gradient across PM

  5. bacterial animal plant Yeast Na+/H+-antiporters Nha1p – PM Kha1p – GA Nhx1p –endosomes ? (Mkh1p)-mitochondria Vnx1p – vacuole ? - ER Přibylová L. et al. Folia Microbiol. 387, 89 (2006)

  6. GeneNHA1 2985 nt, YLR138w, chr XII, low and constitutive expression phenotype:nha1deletion-higher and pH-dependent sensitivity toNa+, Li+, K+, Rb+ - higher pHin NHA1overexpression - higher and pH-dependent tolerance to Na+, Li+, K+, Rb+ activity: Na+, Li+, K+, Rb+ efflux localization: plasma membrane role:detoxification (Na+, Li+) regulation ofintracellular pH K+homeostasis cell volume membrane potential response to osmotic shock

  7. Nha1 protein 985 aa, 110 kDa, 12 tms tms – high similarity with Na+/H+ antiporters from other yeast (Z. rouxii, S. pombe, transpoting only Na+ and Li+) hydrophilic C-terminus 556 aa (56 %)

  8. Detail analysis of region 915-945 aa Growth of buB31 sezkrácenými Nha1p na 30 mM LiCl A Y E S E T E F E R Q R R L N A L G E M T A P A D Q DD EE L

  9. Nha1 protein 985 aa, 110 kDa, 12 tms tms – high similarity with Na+/H+ antiporters from other yeast (Z. rouxii, S. pombe, transpoting only Na+ and Li hydrophilic C-terminus 556 aa (56 %) ●C-terminus is not important for substrate specificity ●Role of C-terminus in K+ homeostasis, regulation of intracellular pH, cell response to osmotic stress, regulation of membrane potential • Is the ScNha1p substrate specificity exceptional?

  10. Sc Nha1 Ca Cnh1 Zr Sod2-22 Sp Sod2 Sc Nha1 100 51.3/ 69.9 / 26.7 57.6 / 76.7 / 34.6 40.8 / 43.0 / 19.6 Ca Cnh1 100 46.1 / 65.5 / 20.0 42.4/ 44.5 / 23.9 Zr Sod2-22 100 41.6 / 43.3 / 23.9 Family of Na+/H+ antiporters in yeast plasma membrane Identity (%) entire sequence / tms + loops / C-terminus

  11. Comparison of yeast plasma-membrane Na+/H+-antiporters C ScNha1p ZrSod2-22 Spsod2 CaCnh1p Substrate specificity Expression from the NHA1 promoter in BW31 cells (ena1-4nha1) Observed tolerance corresponds to antiporters’ transport activity.

  12. Comparison of yeast plasma-membrane Na+/H+-antiporters Z. rouxii ZrSod2-22p andS. pombe sod2pdo not transport K+ and Rb+. S. cerevisiae Nha1p andC. albicans Cnh1phave broad substrate specificity for at least 4 cationts. Other yeast species? • System eliminatingsurplus intracellular K+ inZ. rouxii and S. pombe ?  What determines the antiporter’s substrate specificity?

  13. Yeast with antiporters transporting only Na+, Li+ Yeast with antiporters transporting all alkali metal cations ( Na+, Li+, K+, Rb+) Comparison of yeast plasma-membrane Na+/H+-antiporters Saccharomyces cerevisiae Zygosaccharomyces rouxii Debaryomyces hansenii Pichia sorbitophila Candida albicans Yarrowia lipolytica Schizosaccharomyces pombe Identification of genesin silico, cloning, heterologous expressioninS. cerevisiae, localizationin PM, characterization of substrate specificity

  14. Comparison of yeast plasma-membrane Na+/H+-antiporters Yeast with antiporters transporting only Na+, Li+ Yeast with antiporters transporting all alkali metal cations ( Na+, Li+, K+, Rb+) Saccharomyces cerevisiae (2 systems) Zygosaccharomyces rouxii Debaryomyces hansenii Pichia sorbitophila Candida albicans (2 systems) Yarrowia lipolytica (2 systems) Schizosaccharomyces pombe All yeast species possess a Na+/H+-antiporter regulating[Kin], pHin and cell volume

  15. Amino-acidresiduesdetermining substrate specificity of yeastNa+/H+-antiporters • random mutagenesis (UV, PCR) ofZ. rouxiiSOD2-22 gene (non-transporting K+), expression of mutated versions inS. cerevisiae BW31 (ena1-4∆ nha1∆) • selection of clones growing in the presence of high [KCl], i.e. expresing “K+-transporting“ZrSod2-22p versions

  16. Pro145 Ser150 ZrSod2-22p Thr141 ZrSOD2-22 with mutations in 5th tms S. cerevisiae BW31 (ena1-4∆ nha1∆)

  17. Thr141 Pro145 Ser150 Sequence comparison of 5th tmsof yeast plasma membraneNa+/H+ antiporters

  18. 5th tms Serin or Threonin Pro145 = transport of K+ and Na+ + loss of Li+ transport

  19. 5th tms Ser150 Threonin = transport of K+,Na+ and Li+

  20. 5th tms Thr141 Serin = transport of K+, Na+ and Li+

  21. 5th tms Serin K+ T141, S150 - S141, T150 +/- T141, T150 + S141,S150 + Threonin Side chains of Thr141, Pro145aSer150are involved in conformational changes during binding and/or transport of alkali metal cations via yeast antiporters of the Nha/Sod type

  22. Expression of mammalian Na+/H+ exchangers (NHE) in yeast Protein similarity (%) All tissues Epitel – basolateral side General role, pHin Kidney, small gut (apical side) ? Role, absorption? Kidney, small gut (apical side) ? Role, absorption? Blood pressure

  23. Sensitivity of yeast cells expressing rNHE to alkali metal cations strain BW31 (ena1-4nha1) NHE expression is not toxic NHE2 improves KCl tolerance NHE3 improves NaCl toleranci NHE1 NHE2 NHE3 NHE1 NHE2 NHE3

  24. Growth of yeast cells expressing NHE2p YNB + 800 mM KCl Mammalian antiporters export alkali metal cations in yeast cells

  25. Expression of mKir2.1 channelinS. cerevisiae MAB 2d (ena1-4nha1trk1 trk2) Kir2.1: inward rectified, voltage gated, K+ specific Point mutationsin Kir2.1 genes → activity changes → illness  search for activity modulators Functional expression restore of growth on low [KCl], higher sensitivity to high [KCl]

  26. mKir2.1 expression in MAB2d growth in liquid culture pYEx (control; ∆) pKir2 (♦) 10 mM KCl 100 mM KCl Growth rate depends on mKir2.1 channel activity • Testing activity modulators?

  27. mKir2.1 expression in MAB2d growth in liquid culture  96 cultures (150 l), different [KCl], different modulators

  28. mKir2.1 expression in MAB2d growth in liquid culture

  29. mKir2.1 expression in MAB2d inhibition effect of Ba2+ BaCl2 BW31expressingmKir2.1 () control (pYEx, ) EC50~ 1 mM MAB 2dexpressing mKir2.1 () control (pYEx, ) EC50~ 5 mM  Useful tool for HTS (high throughput screening) of new drugs modulating activity of potassium channels

  30. K+ Ena1-5 Na+ Trk1-2 Mkh1 Na+ K+ H+ Nha1 ? K+ K+ H+ H+ Nhx1 ATP ADP+Pi H+ Tok1 Na+ Vnx1 Na+ K+ K+ H+ K+ Kha1 ADP+Pi ATP NSC1 ? Pma1 H+ Characterization of intracellular antiporters Kha1p – Golgi apparatus, growth at high pHout detoxification of Hygromycin B Nhx1p-endosomes, regulation of cytosolic pH, protein trafficking detoxification of HygromycinB sequestration of Na+ and K+ into vacuoles Strains: Phenotype: LMB01ena1-4∆ nha1∆ kha1∆ no growth at pHout> 7,0 HygS AB11cena1-4∆ nha1∆ nhx1∆ ↑sensitivity to NaCl, KClHygS  expression of plant orthologous genes

  31. GFP-AtChx17p Expression of A. thaliana and O. sativa antiporters in S. cerevisiae StrainAB11cena1-4∆ nha1∆ nhx1∆ OsNhx1p OsNhx1-GFPp NaCl HygB Expression - + +++ AtChx17p Strain LMB01ena1-4∆ nha1∆ kha1∆ pH 4,5 pH 7,0 Expression ScKha1p - AtChx17p • strains with kha1 and nhx1 deletions are suitable for characterization of heterologously expressed transporters from organelles of higher eukaryots

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