The adenine biosynthetic mutants ade1 and ade2 of Saccharomyces cerevisiae accumulate a characteristic red pigment in their vacuoles under adenine limiting conditions. This red pigmentation phenotype, widely used in a variety of genetic screens and assays, is the end product of a glutathione-mediated detoxification pathway, where the glutathione conjugates are transported into the vacuole. The glutathione conjugation step, however, has still remained unsolved. We show here, following a detailed analysis of all the members of the thioredoxin- fold superfamily, the involvement of the monothiol glutaredoxin GRX4 as essential for pigmentation. GRX4 plays multiple roles in the cell, and we show that the role in ade pigmentation does not derive from its regulatory role of the iron transcription factor, Aft1p, but a newly identified GST activity of the protein that we could demonstrate using purified Grx4p. Further, we demonstrate that the GRX domain of GRX4 and its active site cysteine C171 is critical for this activity. The findings thus solve a decades old enigma on a critical step in the formation of this red pigmentation.
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Atkinson HJ and Babbitt PC 2009 Glutathione transferases are structural and functional outliers in the thioredoxin fold. Biochemistry 48 11108–11116
Barbour L and W Xiao 2006 Synthetic lethal screen; in Yeast Protocol (Springer) pp 161–169
Berthelet S, J Usher, K Shulist, A Hamza, N Maltez, et al. 2010 Functional genomics analysis of the Saccharomyces cerevisiae iron responsive transcription factor Aft1 reveals iron-independent functions. Genetics 185 1111–1128
Bharathi V, A Girdhar, A Prasad, M Verma, V Taneja, et al. 2016 Use of ade1 and ade2 mutations for development of a versatile red/white colour assay of amyloid‐induced oxidative stress in saccharomyces cerevisiae. Yeast 33 607–620
Chaudhuri B, Ingavale S and Bachhawat AK 1997 apd1+, a gene required for red pigment formation in ade6 mutants of Schizosaccharomyces pombe, encodes an enzyme required for glutathione biosynthesis: a role for glutathione and a glutathione-conjugate pump. Genetics 145 75–83
Choi JH, Lou W and Vancura A,1998 A novel membrane-bound glutathione S-transferase functions in the stationary phase of the yeast Saccharomyces cerevisiae. J. Biol. Chem. 273 29915–29922
Collinson EJ and Grant CM 2003 Role of yeast glutaredoxins as glutathione S-transferases. J. Biol. Chem. 278 22492–22497
Dorfman B-Z 1969 The isolation of adenylosuccinate synthetase mutants in yeast by selection for constitutive behavior in pigmented strains. Genetics 61 377
Garcerá A, Barreto L, Piedrafita L, Tamarit J and Herrero E 2006 Saccharomyces cerevisiae cells have three Omega class glutathione S-transferases acting as 1-Cys thiol transferases. Biochem. J. 398 187–196
Grant CM 2001 Role of the glutathione/glutaredoxin and thioredoxin systems in yeast growth and response to stress conditions. Mol. Microbiol. 39 533–541
Greetham D, Vickerstaff J, Shenton D, Perrone GG, Dawes IW, et al. 2010 Thioredoxins function as deglutathionylase enzymes in the yeast Saccharomyces cerevisiae. BMC Biochem. 11 3
Gronwald JW and Plaisance KL 1998 Isolation and characterization of glutathione S-transferase isozymes from sorghum. Plant Physiol. 117 877–892
Herrero E and de la Torre-Ruiz MA 2007 Monothiol glutaredoxins: a common domain for multiple functions. Cell. Mol. Life Sci. 64 1518
Ishiguro J 1989 An abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe. Curr. Genet. 15 71–74
Jbel M, Mercier A and Labbé S 2011 Grx4 monothiol glutaredoxin is required for iron limitation-dependent inhibition of Fep1. Eukaryotic Cell 10 629–645
Jones EW and Fink GR 1982 Regulation of amino acid and nucleotide biosynthesis in yeast. Cold Spring Harb. Monogr. Arch. 11 181–299
Kalinina E, Chernov N and Novichkova M 2014 Role of glutathione, glutathione transferase, and glutaredoxin in regulation of redox-dependent processes. Biochemistry 79 1562–1583
Kumar C, Igbaria A, D’autreaux B, Planson AG, Junot C, et al. 2011 Glutathione revisited: a vital function in iron metabolism and ancillary role in thiol‐redox control. EMBO J. 30 2044–2056
Li Z-S, Szczypka M, Lu Y-P, Thiele DJ and Rea PA 1996 The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump. J. Biol. Chem. 271 6509–6517
Ojeda L, Keller G, Muhlenhoff U, Rutherford JC, Lill R, et al. 2006 Role of glutaredoxin-3 and glutaredoxin-4 in the iron regulation of the Aft1 transcriptional activator in Saccharomyces cerevisiae. J. Biol. Chem. 281 17661–17669
Park J, McCormick SP, Cockrell AL, Chakrabarti M and Lindahl PA 2014 High-spin ferric ions in Saccharomyces cerevisiae vacuoles are reduced to the ferrous state during adenine-precursor detoxification. Biochemistry 53 3940–3951
Poulter R and Rikkerink E 1983 Genetic analysis of red, adenine-requiring mutants of Candida albicans. J. Bacteriol. 156 1066–1077
Pujol-Carrion N, Belli G, Herrero E, Nogues A and de la Torre-Ruiz MA 2006 Glutaredoxins Grx3 and Grx4 regulate nuclear localisation of Aft1 and the oxidative stress response in Saccharomyces cerevisiae. J. Cell Sci. 119 4554–4564
Pujol-Carrion N and de la Torre-Ruiz MA 2010 Glutaredoxins Grx4 and Grx3 of Saccharomyces cerevisiae play a role in actin dynamics through their Trx domains, which contributes to oxidative stress resistance. Appl. Environ. Microbiol. 76 7826–7835
Rébora K, Laloo B and Daignan-Fornier B 2005 Revisiting purine-histidine cross-pathway regulation in Saccharomyces cerevisiae: a central role for a small molecule. Genetics 170 61–70
Roman H 1956 A system selective for mutations affecting the synthesis of adenine in yeast. CR Trav. Lab. Carlsberg Ser. Physiol. 26 299–314
Salinas AE and Wong MG 1999 Glutathione S-transferases-a review. Curr. Med. Chem. 6 279–310
Sharma KG, Kaur R and Bachhawat AK 2003 The glutathione-mediated detoxification pathway in yeast: an analysis using the red pigment that accumulates in certain adenine biosynthetic mutants of yeasts reveals the involvement of novel genes. Arch. Microbiol. 180 108–117
Sharma KG, Mason DL, Liu G, Rea PA, Bachhawat AK, et al. 2002 Localization, regulation, and substrate transport properties of Bpt1p, a Saccharomyces cerevisiae MRP-type ABC transporter. Eukaryotic Cell 1 391–400
Vall-llaura N, Reverter-Branchat G, Vived C, Weertman N, Rodríguez-Colman MJ, et al. 2016 Reversible glutathionylation of Sir2 by monothiol glutaredoxins Grx3/4 regulates stress resistance. Free Radical Biol. Med. 96 45–56
Weng Y-S and Nickoloff JA 1997 Nonselective URA3 colony-color assay in yeast ade1 or ade2 mutants. Biotechniques 23 237–242
Yamaguchi‐Iwai Y, Dancis A and Klausner RD 1995 AFT1 a mediator of iron regulated transcriptional control in Saccharomyces cerevisiae. EMBO J. 14 1231–1239
Zonneveld B and van der Zanden A 1995 The red ade mutants of Kluyveromyces lactis and their classification by complementation with cloned ADE1 or ADE2 genes from Saccharomyces cerevisiae. Yeast 11 823–827
This work was funded by a Grant-in-Aid project from the Department of Science and Technology, Government of India to AKB [Grant Number CRG/2018/000190]. AKJ was supported by a DST INSPIRE scholarship, SY was supported by a DST INSPIRE Fellowship [Grant Number (IF150103)]. The authors thank Prarthna for her valuable inputs and support with spotting and figure alignments.
Corresponding editor: BJ Rao
Communicated by BJ Rao.
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Jainarayanan, A.K., Yadav, S. & Bachhawat, A.K. Yeast glutaredoxin, GRX4, functions as a glutathione S-transferase required for red ade pigment formation in Saccharomyces cerevisiae. J Biosci 45, 39 (2020). https://doi.org/10.1007/s12038-020-0015-z
- glutathione conjugation
- glutathione S-transferase
- ade pigmentation