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. 1996 Nov;16(11):6303–6312. doi: 10.1128/mcb.16.11.6303

Suppression of oxidative damage by Saccharomyces cerevisiae ATX2, which encodes a manganese-trafficking protein that localizes to Golgi-like vesicles.

S J Lin 1, V C Culotta 1
PMCID: PMC231633  PMID: 8887660

Abstract

Oxygen toxicity in Saccharomyces cerevisiae lacking the copper/zinc superoxide dismutase (SOD1) can be suppressed by overexpression of the S. cerevisiae ATX2 gene. Multiple copies of ATX2 were found to reverse the aerobic auxotrophies of sod1(delta) mutants for lysine and methionine and also to enhance the resistance of these yeast strains to paraquat and atmospheric levels of oxygen. ATX2 encodes a novel 34.4-kDa polypeptide with a number of potential membrane-spanning domains. Our studies indicate that Atx2p localizes to the membrane of a vesicular compartment in yeast cells reminiscent of the Golgi apparatus. With indirect immunofluorescence microscopy, Atx2p exhibited a punctate pattern of staining typical of the Golgi apparatus, and upon subcellular fractionation, Atx2p colocalized with a biochemical marker for the yeast Golgi apparatus. We demonstrate here that this vesicle protein normally functions in the homeostasis of manganese ions and that this role in metal metabolism is necessary for the ATX1 suppression of SOD1 deficiency. First, overexpression of ATX2 caused cells to accumulate increased levels of manganese. Second, a deletion in ATX2 caused a decrease in the apparent available level of intracellular manganese and caused sod1(delta) mutants to become dependent upon exogenous manganese for aerobic growth. Third, ATX2 was incapable of suppressing oxidative damage in cells depleted of manganese ions or lacking the plasma membrane transporter for manganese. The effect of ATX2 overexpression on manganese accumulation and oxygen resistance is similar to what we have previously reported for mutations in PMR1, which encodes a manganese-trafficking protein that also resides in a vesicular compartment. Our studies are consistent with a model in which Atx2p and Pmr1p work in opposite directions to control manganese homeostasis.

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Selected References

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  1. Antebi A., Fink G. R. The yeast Ca(2+)-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution. Mol Biol Cell. 1992 Jun;3(6):633–654. doi: 10.1091/mbc.3.6.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Archibald F. S., Fridovich I. Manganese and defenses against oxygen toxicity in Lactobacillus plantarum. J Bacteriol. 1981 Jan;145(1):442–451. doi: 10.1128/jb.145.1.442-451.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Archibald F. S., Fridovich I. The scavenging of superoxide radical by manganous complexes: in vitro. Arch Biochem Biophys. 1982 Apr 1;214(2):452–463. doi: 10.1016/0003-9861(82)90049-2. [DOI] [PubMed] [Google Scholar]
  4. Becker D. M., Guarente L. High-efficiency transformation of yeast by electroporation. Methods Enzymol. 1991;194:182–187. doi: 10.1016/0076-6879(91)94015-5. [DOI] [PubMed] [Google Scholar]
  5. Berkower C., Loayza D., Michaelis S. Metabolic instability and constitutive endocytosis of STE6, the a-factor transporter of Saccharomyces cerevisiae. Mol Biol Cell. 1994 Nov;5(11):1185–1198. doi: 10.1091/mbc.5.11.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bermingham-McDonogh O., Gralla E. B., Valentine J. S. The copper, zinc-superoxide dismutase gene of Saccharomyces cerevisiae: cloning, sequencing, and biological activity. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4789–4793. doi: 10.1073/pnas.85.13.4789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Biliński T., Krawiec Z., Liczmański A., Litwińska J. Is hydroxyl radical generated by the Fenton reaction in vivo? Biochem Biophys Res Commun. 1985 Jul 31;130(2):533–539. doi: 10.1016/0006-291x(85)90449-8. [DOI] [PubMed] [Google Scholar]
  8. Bostek C. C. Oxygen toxicity: an introduction. AANA J. 1989 Jun;57(3):231–237. [PubMed] [Google Scholar]
  9. Carlioz A., Touati D. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 1986 Mar;5(3):623–630. doi: 10.1002/j.1460-2075.1986.tb04256.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chang E. C., Kosman D. J. Intracellular Mn (II)-associated superoxide scavenging activity protects Cu,Zn superoxide dismutase-deficient Saccharomyces cerevisiae against dioxygen stress. J Biol Chem. 1989 Jul 25;264(21):12172–12178. [PubMed] [Google Scholar]
  11. Chang E. C., Kosman D. J. O2-dependent methionine auxotrophy in Cu,Zn superoxide dismutase-deficient mutants of Saccharomyces cerevisiae. J Bacteriol. 1990 Apr;172(4):1840–1845. doi: 10.1128/jb.172.4.1840-1845.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Culotta V. C., Howard W. R., Liu X. F. CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae. J Biol Chem. 1994 Oct 14;269(41):25295–25302. [PubMed] [Google Scholar]
  13. Culotta V. C., Joh H. D., Lin S. J., Slekar K. H., Strain J. A physiological role for Saccharomyces cerevisiae copper/zinc superoxide dismutase in copper buffering. J Biol Chem. 1995 Dec 15;270(50):29991–29997. doi: 10.1074/jbc.270.50.29991. [DOI] [PubMed] [Google Scholar]
  14. Cunningham K. W., Fink G. R. Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases. J Cell Biol. 1994 Feb;124(3):351–363. doi: 10.1083/jcb.124.3.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dancis A., Klausner R. D., Hinnebusch A. G., Barriocanal J. G. Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae. Mol Cell Biol. 1990 May;10(5):2294–2301. doi: 10.1128/mcb.10.5.2294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Deng H. X., Hentati A., Tainer J. A., Iqbal Z., Cayabyab A., Hung W. Y., Getzoff E. D., Hu P., Herzfeldt B., Roos R. P. Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. Science. 1993 Aug 20;261(5124):1047–1051. doi: 10.1126/science.8351519. [DOI] [PubMed] [Google Scholar]
  17. Farr S. B., D'Ari R., Touati D. Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8268–8272. doi: 10.1073/pnas.83.21.8268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Faulkner K. M., Liochev S. I., Fridovich I. Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo. J Biol Chem. 1994 Sep 23;269(38):23471–23476. [PubMed] [Google Scholar]
  19. Feldheim D., Rothblatt J., Schekman R. Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation. Mol Cell Biol. 1992 Jul;12(7):3288–3296. doi: 10.1128/mcb.12.7.3288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Franzusoff A., Redding K., Crosby J., Fuller R. S., Schekman R. Localization of components involved in protein transport and processing through the yeast Golgi apparatus. J Cell Biol. 1991 Jan;112(1):27–37. doi: 10.1083/jcb.112.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fridovich I. Superoxide dismutases. Adv Enzymol Relat Areas Mol Biol. 1986;58:61–97. doi: 10.1002/9780470123041.ch2. [DOI] [PubMed] [Google Scholar]
  22. Fridovich I. Superoxide radical: an endogenous toxicant. Annu Rev Pharmacol Toxicol. 1983;23:239–257. doi: 10.1146/annurev.pa.23.040183.001323. [DOI] [PubMed] [Google Scholar]
  23. Fridovich I. The biology of oxygen radicals. Science. 1978 Sep 8;201(4359):875–880. doi: 10.1126/science.210504. [DOI] [PubMed] [Google Scholar]
  24. Galiazzo F., Schiesser A., Rotilio G. Oxygen-independent induction of enzyme activities related to oxygen metabolism in yeast by copper. Biochim Biophys Acta. 1988 Apr 14;965(1):46–51. doi: 10.1016/0304-4165(88)90149-3. [DOI] [PubMed] [Google Scholar]
  25. Gralla E. B., Kosman D. J. Molecular genetics of superoxide dismutases in yeasts and related fungi. Adv Genet. 1992;30:251–319. doi: 10.1016/s0065-2660(08)60322-3. [DOI] [PubMed] [Google Scholar]
  26. Gralla E. B., Valentine J. S. Null mutants of Saccharomyces cerevisiae Cu,Zn superoxide dismutase: characterization and spontaneous mutation rates. J Bacteriol. 1991 Sep;173(18):5918–5920. doi: 10.1128/jb.173.18.5918-5920.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Haselbeck A., Schekman R. Interorganelle transfer and glycosylation of yeast invertase in vitro. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2017–2021. doi: 10.1073/pnas.83.7.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  29. Lamb J. R., Michaud W. A., Sikorski R. S., Hieter P. A. Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis. EMBO J. 1994 Sep 15;13(18):4321–4328. doi: 10.1002/j.1460-2075.1994.tb06752.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lapinskas P. J., Cunningham K. W., Liu X. F., Fink G. R., Culotta V. C. Mutations in PMR1 suppress oxidative damage in yeast cells lacking superoxide dismutase. Mol Cell Biol. 1995 Mar;15(3):1382–1388. doi: 10.1128/mcb.15.3.1382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lapinskas P. J., Lin S. J., Culotta V. C. The role of the Saccharomyces cerevisiae CCC1 gene in the homeostasis of manganese ions. Mol Microbiol. 1996 Aug;21(3):519–528. doi: 10.1111/j.1365-2958.1996.tb02561.x. [DOI] [PubMed] [Google Scholar]
  32. Lee K. S., Patton J. L., Fido M., Hines L. K., Kohlwein S. D., Paltauf F., Henry S. A., Levin D. E. The Saccharomyces cerevisiae PLB1 gene encodes a protein required for lysophospholipase and phospholipase B activity. J Biol Chem. 1994 Aug 5;269(31):19725–19730. [PubMed] [Google Scholar]
  33. Lin S. J., Culotta V. C. The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3784–3788. doi: 10.1073/pnas.92.9.3784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liu X. F., Culotta V. C. The requirement for yeast superoxide dismutase is bypassed through mutations in BSD2, a novel metal homeostasis gene. Mol Cell Biol. 1994 Nov;14(11):7037–7045. doi: 10.1128/mcb.14.11.7037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Liu X. F., Elashvili I., Gralla E. B., Valentine J. S., Lapinskas P., Culotta V. C. Yeast lacking superoxide dismutase. Isolation of genetic suppressors. J Biol Chem. 1992 Sep 15;267(26):18298–18302. [PubMed] [Google Scholar]
  36. Raths S., Rohrer J., Crausaz F., Riezman H. end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J Cell Biol. 1993 Jan;120(1):55–65. doi: 10.1083/jcb.120.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Riles L., Dutchik J. E., Baktha A., McCauley B. K., Thayer E. C., Leckie M. P., Braden V. V., Depke J. E., Olson M. V. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. doi: 10.1093/genetics/134.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rosen D. R., Siddique T., Patterson D., Figlewicz D. A., Sapp P., Hentati A., Donaldson D., Goto J., O'Regan J. P., Deng H. X. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993 Mar 4;362(6415):59–62. doi: 10.1038/362059a0. [DOI] [PubMed] [Google Scholar]
  39. Rudolph H. K., Antebi A., Fink G. R., Buckley C. M., Dorman T. E., LeVitre J., Davidow L. S., Mao J. I., Moir D. T. The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family. Cell. 1989 Jul 14;58(1):133–145. doi: 10.1016/0092-8674(89)90410-8. [DOI] [PubMed] [Google Scholar]
  40. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Supek F., Supekova L., Nelson H., Nelson N. A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria. Proc Natl Acad Sci U S A. 1996 May 14;93(10):5105–5110. doi: 10.1073/pnas.93.10.5105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tamai K. T., Gralla E. B., Ellerby L. M., Valentine J. S., Thiele D. J. Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8013–8017. doi: 10.1073/pnas.90.17.8013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  44. van Loon A. P., Pesold-Hurt B., Schatz G. A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3820–3824. doi: 10.1073/pnas.83.11.3820. [DOI] [PMC free article] [PubMed] [Google Scholar]

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