Current Genetics

, Volume 49, Issue 1, pp 7–20 | Cite as

Residues of the yeast ALR1 protein that are critical for Magnesium uptake

Research Article

Abstract

Mutagenesis was used to study the function by the ALR1 (aluminium resistance) gene, which encodes the major Mg2+ uptake system in yeast. Truncation of Alr1 showed that the N-terminal 239 amino acids and the C-terminal 53 amino acids are not essential for magnesium uptake. Random PCR mutagenesis was undertaken of the C-terminal part of ALR1 that is homologous to the bacterial CorA magnesium transport family. The mutants with the most severe phenotype all had amino acid changes in a small region containing the putative transmembrane domains. Eighteen single amino acid mutants in this critical region were classified into three categories for magnesium uptake: no, low and moderate activity. Seventeen of the 18 mutants expressed a cross-reacting band of similar size and intensity as wild-type Alr1. Conservative mutations that reduced or inactivated uptake led us to identify Ser729, Ile746 and Met762 (part of the conserved GMN motif) as critical amino acid residues in Alr1. High expression of inactive mutants inhibited the capability of wild-type Alr1 to transport magnesium, consistent with Alr1 forming homo-oligomers. The results confirm the classification of ALR1 as a member of the CorA family of magnesium transport genes.

Keywords

Saccharomyces cerevisiae PCR mutagenesis Magnesium CorA gene family Transmembrane domains Dominant negative Atomic absorption spectrophotometry 

Abbreviations

TM

transmembrane

YPD

yeast extract peptone dextrose

YPDM

YPD plus high magnesium

LPM

low pH low magnesium

SC

synthetic complete

OD

Optical density

HA

Haemaglutenin

ER

endoplasmic reticulum

PCR

Polymerase chain reaction

References

  1. Bonneaud N, Ozierkalogeropoulos O, Li GY, Labouesse M, Minviellesebastia L, Lacroute F (1991) A family of low and high copy replicative, integrative and single-stranded Saccharomyces-cerevisiae Escherichia-coli shuttle vectors. Yeast 7:609–615PubMedCrossRefGoogle Scholar
  2. Bui DM, Gregan J, Jarosch E, Ragnini A, Schweyen RJ (1999) The bacterial magnesium transporter CorA can functionally substitute for its putative homologue Mrs2p in the yeast inner mitochondrial membrane. J Biol Chem 274:20438–20443PubMedCrossRefGoogle Scholar
  3. Caldwell AM, Smith RL (2003) Membrane topology of the ZntB efflux system of Salmonella enterica Serovar Typhimurium. J Bacteriol 185:374–376PubMedCrossRefGoogle Scholar
  4. Casagrande R, Stern P, Diehn M, Shamu C, Osario M, Zuniga M, Brown PO, Ploegh H (2000) Degradation of proteins from the ER of S. cerevisiae requires an intact unfolded protein response pathway. Mol Cell 5:729–735PubMedCrossRefGoogle Scholar
  5. Chang G, Spencer RH, Lee AT, Barclay MT, Rees DC (1998) Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel. Science 282:2220–2226PubMedCrossRefGoogle Scholar
  6. Doyle DA, Cabral JaoM, Pfuetzner RA, Kuo A, Gulbis JM, Cohen SL, Chait BT, MacKinnon R (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science 280:69–77PubMedCrossRefGoogle Scholar
  7. Drummond RSM, Tutone A, Li Y-C, Gardner RC (2005) A putative magnesium transporter AtMRS2–11 is localized to the chloroplastic envelope membrane. Plant Sci (in press)Google Scholar
  8. Ezaki B, Sivaguru M, Ezaki Y, Matsumoto H, Gardner RC (1999) Acquisition of aluminum tolerance in Saccharomyces cerevisiae by expression of the BCB or NtGDI1 gene derived from plants. FEMS Microbiol Lett 171:81–87PubMedCrossRefGoogle Scholar
  9. Ezquerra M, Carnero C, Blesa R, Gelpi JL, Ballesta F, Oliva R (1999) A presenilin 1 mutation (Ser169Pro) associated with early-onset AD and myoclonic seizures. Neurology 52:566–570PubMedGoogle Scholar
  10. Fromant M, Blanquet S, Plateau P (1995) Direct random mutagenesis of gene-sized DNA fragments using polymerase chain-reaction. Anal Biochem 224:347–353PubMedCrossRefGoogle Scholar
  11. Fuhrmann GF, Rothstein A (1968) The transport of Zn2+, Co2+ and Ni2+ into yeast cells. Biochim Biophys Acta 163:325–330PubMedCrossRefGoogle Scholar
  12. Gardner RC (2003) Genes for magnesium transport. Curr Opin Plant Biol 6:263–267PubMedCrossRefGoogle Scholar
  13. Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360PubMedCrossRefGoogle Scholar
  14. Graschopf A, Stadler JA, Hoellerer MK, Eder S, Sieghardt M, Kohlwein SD, Schweyen RJ (2001) The yeast plasma membrane protein Alr1 controls Mg2+ homeostasis and is subject to Mg2+-dependent control of its synthesis and degradation. J Biol Chem 276:16216–16222PubMedCrossRefGoogle Scholar
  15. Grubbs RD, Snavely MD, Paul Hmiel S, Maguire ME (1989) [36] Magnesium transport in eukaryotic and prokaryotic cells using magnesium-28 ion. Methods in enzymology, vol 173. Academic, New York, pp 546–563Google Scholar
  16. Hmiel SP, Snavely MD, Miller CG, Maguire ME (1986) Magnesium transport in Salmonella typhimurium: characterization of magnesium influx and cloning of a transport gene. J Bacteriol 168:1444–1450PubMedGoogle Scholar
  17. Kehres DG, Lawyer CH, Maguire ME (1998) The CorA magnesium transporter gene family. Microb Comp Genomics 3:151–169PubMedGoogle Scholar
  18. Kehres DG, Maguire ME (2002) Structure, properties and regulation of magnesium transport proteins. BioMetals 15:261–270PubMedCrossRefGoogle Scholar
  19. Kolisek M, Zsurka G, Samaj J, Weghuber J, Schweyen RJ, Schweigel M (2003) Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria. EMBO J 22:1235–1244CrossRefPubMedGoogle Scholar
  20. Kuo A, Gulbis JM, Antcliff JF, Rahman T, Lowe ED, Zimmer J, Cuthbertson J, Ashcroft FM, Ezaki T, Doyle DA (2003) Crystal structure of the potassium channel KirBac1.1 in the closed state. Science 300:1922–1926CrossRefPubMedGoogle Scholar
  21. Kushnirov VV (2000) Rapid and reliable protein extraction from yeast. Yeast 16:857–860PubMedCrossRefGoogle Scholar
  22. Lee J, Pena MMO, Nose Y, Thiele DJ (2002) Biochemical characterization of the human copper transporter Ctr1. J Biol Chem 277:4380–4387PubMedCrossRefGoogle Scholar
  23. Lee K, Neigeborn L, Kaufman RJ (2003) The unfolded protein response is required for haploid tolerance in yeast. J Biol Chem 278:11818–11827PubMedCrossRefGoogle Scholar
  24. Li L, Tutone AF, Drummond RS, Gardner RC, Luan S (2001) A novel family of magnesium transport genes in Arabidopsis. Plant Cell 13:2761–2775PubMedCrossRefGoogle Scholar
  25. Liu GJ, Martin DK, Gardner RC, Ryan PR (2002) Large Mg(2+)-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae. FEMS Microbiol Lett 213:231–237PubMedCrossRefGoogle Scholar
  26. Longtine MS, McKenzie A, III, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953–961PubMedCrossRefGoogle Scholar
  27. MacDiarmid CW, Gardner RC (1996) A1 toxicity in yeast. A role for Mg? Plant Physiol 112:1101–1109Google Scholar
  28. MacDiarmid CW, Gardner RC (1998) Overexpression of the Saccharomyces cerevisiae magnesium transport system confers resistance to aluminum ion. J Biol Chem 273:1727–1732CrossRefPubMedGoogle Scholar
  29. Marini AM, Springael JY, Frommer WB, Andre B (2000) Cross-talk between ammonium transporters in yeast and interference by the soybean SAT1 protein. Mol Microbiol 35:378–385PubMedCrossRefGoogle Scholar
  30. Monahan BJ, Unkles SE, Tsing IT, Kinghorn JR, Hynes MJ, Davis MA (2002) Mutation and functional analysis of the Aspergillus nidulans ammonium permease MeaA and evidence for interaction with itself and MepA. Fungal Genet Biol 36:35–46PubMedCrossRefGoogle Scholar
  31. Muhlrad D, Hunter R, Parker R (1992) A rapid method for localized mutagenesis of yeast genes. Yeast 8:79–82PubMedCrossRefGoogle Scholar
  32. Patil C, Walter P (2001) Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. Curr Opin Cell Biol 13:349–355PubMedCrossRefGoogle Scholar
  33. Pena MMO, Puig S, Thiele DJ (2000) Characterization of the Saccharomyces cerevisiae high affinity copper transporter Ctr3. J Biol Chem 275:33244–33251PubMedCrossRefGoogle Scholar
  34. Schock I, Gregan J, Steinhauser S, Schweyen R, Brennicke A, Knoop V (2000) A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant. Plant J 24:489–501PubMedCrossRefGoogle Scholar
  35. Serrano R, Monk BC, Villalba JM, Montesinos C, Weiler EW (1993) Epitope mapping and accessibility of immunodominant regions of yeast plasma membrane H(+)-ATPase. Eur J Biochem 212:737–744PubMedCrossRefGoogle Scholar
  36. Sherman F (1991) Getting started with yeast. Methods Enzymol 194:3–21PubMedCrossRefGoogle Scholar
  37. Smith JA, Pease LG (1980) Reverse turns in peptides and proteins. Crc Crit Rev Biochem 8:315–399PubMedCrossRefGoogle Scholar
  38. Smith RL, Banks JL, Snavely MD, Maguire ME (1993) Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coli. Identification of a new class of transport protein. J Biol Chem 268:14071–14080PubMedGoogle Scholar
  39. Smith RL, Szegedy MA, Kucharski LM, Walker C, Wiet RM, Redpath A, Kaczmarek MT, Maguire ME (1998) The CorA Mg2+ transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the third membrane domain identifies a Mg2+ pore. J Biol Chem 273:28663–28669PubMedCrossRefGoogle Scholar
  40. Svetlov V, Cooper TG (1998) Efficient PCR-based random mutagenesis of sub-genic (100 bp) DNA fragments. Yeast 14:89–91CrossRefPubMedGoogle Scholar
  41. Szegedy MA, Maguire ME (1999) The CorA Mg(2+) transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the second membrane domain. J Biol Chem 274:36973–36979CrossRefPubMedGoogle Scholar
  42. Warren MA, Kucharski LM, Veenstra A, Shi L, Grulich PF, Maguire ME (2004) The CorA Mg2+ transporter is a homotetramer. J Bacteriol 186:4605–4612PubMedCrossRefGoogle Scholar
  43. Worlock AJ, Smith RL (2002) ZntB is a novel Zn2+ transporter in Salmonella enterica serovar Typhimurium. J Bacteriol 184:4369–4373CrossRefPubMedGoogle Scholar
  44. Yerushalmi H, Lebendiker M, Schuldiner S (1996) Negative dominance studies demonstrate the oligomeric structure of EmrE, a multidrug antiporter from Escherichia coli. J Biol Chem 271:31044–31048PubMedCrossRefGoogle Scholar
  45. Zsurka G, Gregan J, Schweyen RJ (2001) The human mitochondrial Mrs2 protein functionally substitutes for its yeast homologue, a candidate magnesium transporter. Genomics 72:158–168PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.School of Biological SciencesUniversity of AucklandAucklandNew Zealand

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