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- W1993669834 abstract "Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family. In bacterial, yeast, and mammalian cells, Grxs appear to be involved in maintaining cellular redox homeostasis. However, in plants, the physiological roles of Grxs have not been fully characterized. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified but not well characterized. Here we demonstrate that a plant protein, AtGRXcp, is a chloroplast-localized monothiol Grx with high similarity to yeast Grx5. In yeast expression assays, AtGRXcp localized to the mitochondria and suppressed the sensitivity of yeast grx5 cells to H2O2 and protein oxidation. AtGRXcp expression can also suppress iron accumulation and partially rescue the lysine auxotrophy of yeast grx5 cells. Analysis of the conserved monothiol motif suggests that the cysteine residue affects AtGRXcp expression and stability. In planta, AtGRXcp expression was elevated in young cotyledons, green tissues, and vascular bundles. Analysis of atgrxcp plants demonstrated defects in early seedling growth under oxidative stresses. In addition, atgrxcp lines displayed increased protein carbonylation within chloroplasts. Thus, this work describes the initial functional characterization of a plant monothiol Grx and suggests a conserved biological function in protecting cells against protein oxidative damage. Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family. In bacterial, yeast, and mammalian cells, Grxs appear to be involved in maintaining cellular redox homeostasis. However, in plants, the physiological roles of Grxs have not been fully characterized. Recently, an emerging subgroup of Grxs with one cysteine residue in the putative active motif (monothiol Grxs) has been identified but not well characterized. Here we demonstrate that a plant protein, AtGRXcp, is a chloroplast-localized monothiol Grx with high similarity to yeast Grx5. In yeast expression assays, AtGRXcp localized to the mitochondria and suppressed the sensitivity of yeast grx5 cells to H2O2 and protein oxidation. AtGRXcp expression can also suppress iron accumulation and partially rescue the lysine auxotrophy of yeast grx5 cells. Analysis of the conserved monothiol motif suggests that the cysteine residue affects AtGRXcp expression and stability. In planta, AtGRXcp expression was elevated in young cotyledons, green tissues, and vascular bundles. Analysis of atgrxcp plants demonstrated defects in early seedling growth under oxidative stresses. In addition, atgrxcp lines displayed increased protein carbonylation within chloroplasts. Thus, this work describes the initial functional characterization of a plant monothiol Grx and suggests a conserved biological function in protecting cells against protein oxidative damage. Reactive oxygen species (ROS) 2The abbreviations used are: ROS, reactive oxygen species; PICOT-HD, protein kinase C-interacting cousin of thioredoxin homology domain; Grx, glutaredoxin; Trx, thioredoxin; GFP, green fluorescent protein; GUS, β-glucuronidase gene; CaMV, cauliflower mosaic virus promoter.2The abbreviations used are: ROS, reactive oxygen species; PICOT-HD, protein kinase C-interacting cousin of thioredoxin homology domain; Grx, glutaredoxin; Trx, thioredoxin; GFP, green fluorescent protein; GUS, β-glucuronidase gene; CaMV, cauliflower mosaic virus promoter. can be formed as by-products in all oxygenic organisms during aerobic metabolism (1Apel K. Hirt H. Annu. Rev. Plant Biol. 2004; 55: 373-399Crossref PubMed Scopus (7919) Google Scholar). In higher plants, chloroplasts and mitochondria are two major organelles that contribute to production of reactive oxygen species during photosynthesis and carbon metabolism (2Vranová E. Inzé D. Van Breusegem F. J. Exp. Bot. 2002; 53: 1227-1236Crossref PubMed Google Scholar, 3Rhoads D.M. Umbach A.L. Chalivendra S.C. Siedow J.N. Plant Physiol. 2006; 141: 357-366Crossref PubMed Scopus (386) Google Scholar). In addition, plants actively generate ROS as signals in response to environmental stresses (3Rhoads D.M. Umbach A.L. Chalivendra S.C. Siedow J.N. Plant Physiol. 2006; 141: 357-366Crossref PubMed Scopus (386) Google Scholar, 4Foyer C.H. Noctor G. Plant Cell. 2005; 17: 1866-1875Crossref PubMed Scopus (2024) Google Scholar, 5Fobert P.R. Després C. Curr. Opin. Plant Biol. 2005; 8: 378-382Crossref PubMed Scopus (115) Google Scholar, 6Gechev T.S. Hille J. J. Cell Biol. 2005; 168: 17-20Crossref PubMed Scopus (386) Google Scholar). However, because of the cytotoxic and extremely reactive nature of ROS, they can cause wide ranging damage to macromolecules (1Apel K. Hirt H. Annu. Rev. Plant Biol. 2004; 55: 373-399Crossref PubMed Scopus (7919) Google Scholar, 7Finkel T. Holbrook N.J. Nature. 2000; 408: 239-247Crossref PubMed Scopus (7200) Google Scholar, 8Nyström T. EMBO J. 2005; 24: 1311-1317Crossref PubMed Scopus (599) Google Scholar, 9Johansson E. Olsson O. Nyström T. J. Biol. Chem. 2004; 279: 22204-22208Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). To overcome such oxidative damage and control signaling events, plants have orchestrated an elaborate antioxidant network (4Foyer C.H. Noctor G. Plant Cell. 2005; 17: 1866-1875Crossref PubMed Scopus (2024) Google Scholar).Of those antioxidant systems, the physiological roles of thioredoxins have been intensively studied (10Buchanan B.B. Balmer Y. Annu. Rev. Plant Biol. 2005; 56: 187-220Crossref PubMed Scopus (689) Google Scholar), whereas those of Grxs have not been fully defined (11Fernandes A.P. Holmgren A. Antioxid. Redox Signal. 2004; 6: 63-74Crossref PubMed Scopus (534) Google Scholar, 12Rouhier N. Gelhaye E. Jacquot J.-P. Cell Mol. Life. Sci. 2004; 61: 1266-1277Crossref PubMed Scopus (169) Google Scholar). Grxs are ubiquitous small heat-stable disulfide oxidoreductases, which are conserved in both prokaryotes and eukaryotes (11Fernandes A.P. Holmgren A. Antioxid. Redox Signal. 2004; 6: 63-74Crossref PubMed Scopus (534) Google Scholar, 13Holmgren A. Åslund F. Methods Enzymol. 1995; 252: 283-292Crossref PubMed Scopus (296) Google Scholar). Through an active motif, namely the conserved CPYC sequence (a dithiol Grx), they catalyze the reduction of protein disulfides and of GSH-protein mixed disulfides via a dithiol or monothiol mechanism (14Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 15Bushweller J.H. Åslund F. Wüthrich K. Holmgren A. Biochemistry. 1992; 31: 9288-9293Crossref PubMed Scopus (203) Google Scholar). In bacterial, yeast, and mammalian cells, dithiol Grxs appear to be involved in many cellular processes and play an important role in protecting cells against oxidative stresses (16Ortenberg R. Gon S. Porat A. Beckwith J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7439-7444Crossref PubMed Scopus (55) Google Scholar, 17Grant C.M. Mol. Microbiol. 2001; 39: 533-541Crossref PubMed Scopus (327) Google Scholar, 18Lillig C.H. Berndt C. Vergnolle O. Lönn M.E. Hudemann C. Bill E. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8168-8173Crossref PubMed Scopus (235) Google Scholar).Besides the dithiol Grxs, a new group of monothiol Grxs has recently been identified in yeast (Grx3, -4, and -5) and bacteria (Grx4) that have a single cysteine residue in the putative active motif (19Rodrígues-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (263) Google Scholar, 20Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; 280: 24544-24552Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Yeast Grx5 encodes a mitochondrial monothiol Grx, which is required for biogenesis of iron-sulfur clusters, whereas Grx3 and Grx4 function in detoxification of cytotoxin and cell proliferation in yeast (21Rodríguez-Manzaneque M.T. Tamarit J. Bellí G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (390) Google Scholar, 22Jablonowski D. Butler A.R. Fichtner L. Gardiner D. Schaffrath R. Stark M.J.R. Genetics. 2001; 159: 1479-1489Crossref PubMed Google Scholar, 23Lopreiato R. Facchin S. Sartori G. Arrigoni G. Casonato S. Ruzzene M. Pinna L.A. Carignani G. Biochem. J. 2004; 377: 395-405Crossref PubMed Scopus (58) Google Scholar). Interestingly, bacterial Grx4, unlike other previously characterized Grxs, can serve as a substrate for thioredoxin reductase instead of NADPH/glutathione reductase (20Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; 280: 24544-24552Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), suggesting that those monothiol Grxs have distinct functions. This group of monothiol Grxs is also conserved across organisms and has now been identified in malarial parasites, plants, zebrafish, mice, and humans (24Witte S. Villalba M. Bi K. Liu Y. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 25Rahlfs S. Fischer M. Becker K. J. Biol. Chem. 2001; 276: 37133-37140Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 26Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2003; 278: 6503-6509Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 27Wingert R.A. Galloway J.L. Barut B. Foott H. Fraenkel P. Axe J.L. Weber G.J. Dooley K. Davidson A.J. Schmid B. et al.Nature. 2005; 436: 1035-1039Crossref PubMed Scopus (320) Google Scholar). Recent studies also indicate that those Grxs contain a newly identified protein kinase C-interacting cousin of thioredoxin homology domain (PICOT-HD) in their carboxyl-terminal regions (24Witte S. Villalba M. Bi K. Liu Y. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 28Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, ascertaining a unifying function of PICOT-HD Grxs has been problematic.Photosynthetic organisms, particularly higher plants, have a large Grx gene family; however, until recently, only a few plant Grxs have been studied (12Rouhier N. Gelhaye E. Jacquot J.-P. Cell Mol. Life. Sci. 2004; 61: 1266-1277Crossref PubMed Scopus (169) Google Scholar, 29Rouhier N. Vlamis-Gardikas A. Lillig C.H. Berndt C. Schwenn J.-D. Holmgren A. Jacquot J.-P. Antioxid. Redox Signal. 2003; 5: 15-22Crossref PubMed Scopus (35) Google Scholar, 30Lemaire S.D. Photosynth. Res. 2004; 79: 305-318Crossref PubMed Scopus (119) Google Scholar). In the Arabidopsis genome, there are at least 31 open reading frames coding for putative Grxs, which can be divided into three major classes that include the aforementioned monothiol Grxs (12Rouhier N. Gelhaye E. Jacquot J.-P. Cell Mol. Life. Sci. 2004; 61: 1266-1277Crossref PubMed Scopus (169) Google Scholar, 30Lemaire S.D. Photosynth. Res. 2004; 79: 305-318Crossref PubMed Scopus (119) Google Scholar). Genetic analysis of a CC type Grx, ROXY1, indicates an important role of this protein in floral petal development (31Xing S. Rosso M.G. Zachgo S. Development. 2005; 132: 1555-1565Crossref PubMed Scopus (127) Google Scholar). In a previous study, we used a yeast functional screen to identify a PICOT-HD-containing protein that was able to activate Arabidopsis CAX1 Ca2+/H+ antiport activity in a yeast expression system; this gene was originally termed CXIP1 (26Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2003; 278: 6503-6509Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Here we determine the subcellular localization of this first cloned plant PICOT-HD-containing protein and reclassify the gene as AtGRXcp. We functionally characterize the protein and perform initial structure-function studies using yeast expression assays. We go on to isolate AtGRXcp knock-out mutants and describe the phenotypes of these plants at the whole plant and biochemical levels. For the first time, we demonstrate a role for a plant monothiol Grx.EXPERIMENTAL PROCEDURESIsolation of AtGRXcp Null Alleles—To isolate atgrxcp alleles, two T-DNA insertional mutant lines were obtained from the SALK T-DNA collection (32Alonso J.M. Stepanova A.N. Leisse T.J. Kim C.J. Chen H. Shinn P. Stevenson D.K. Zimmerman J. Barajas P. Cheuk R. et al.Science. 2003; 301: 653-657Crossref PubMed Scopus (4087) Google Scholar). Homozygous plants from each T3 generation were obtained by PCR screening using AtGRXcp-specific and T-DNA border primers. An AtGRXcp reverse primer, 5′-GGG CCG GAT CCT CGA GTC AAG AGC ACA TAG CTT TCT C-3′, and a T-DNA left border primer, 5′-GCG TGG ACC GCT TGC TGC A-3′, were used to screen for the atgrxcp1 allele. The AtGRXcp reverse primer and a T-DNA right border primer, 5′-TGG GAA AAC CTG GCG TTA CCC AAC TTA AT-3′, were used to screen for the atgrxcp2 allele. An AtGRXcp forward primer, 5′-GGC AAG CTT ATA AGT TTT AAT CGT TTA TGG GGT-3′, and the AtGRXcp reverse primer were used to amplify the wild type AtGRXcp gene. The location of the T-DNA insertion was determined by sequencing the PCR product. Both atgrxcp alleles were back-crossed to their respective parental plants to remove any potential unlinked mutations.Plant Growth Conditions—Arabidopsis wild type (ecotype Columbia, Col-0) and atgrxcp mutant seeds were surface-sterilized, germinated, and grown on one-half strength Murashige and Skoog medium (33Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (53131) Google Scholar) solidified with 0.8% agar and the same medium supplemented with various concentrations of H2O2. Iron-sufficient and -deficient media were made following a published protocol (34Vert G. Grotz N. Dédaldéchamp F. Gaymard F. Guerinot M.L. Briat J.-F. Curie C. Plant Cell. 2002; 14: 1223-1233Crossref PubMed Scopus (1119) Google Scholar).DNA Constructs and Site-directed Mutagenesis—Yeast Grx5 was amplified by PCR using a forward primer (5′-GCC GGA TCC ATG TTT CTC CCA AAA TTC AAT-3′) and a reverse primer (5′-CCG GAG CTC TCA ACG ATC TTT GGT TTC TTC-3′), and the PCR products were cloned into pGEM-T Easy (Promega, Madison, WI). The full-length cDNA of AtGRXcp was isolated through a yeast functional screen and originally termed CXIP1 (for CAX1-interacting protein 1) (26Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2003; 278: 6503-6509Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). AtGRXcp was predicted to have a 63-amino acid signal peptide by analysis with the Chloro P (version 1.1) program (available on the World Wide Web at www.cbs.dtu.dk/services/ChloroP/). To remove this N-terminal signal peptide, a truncated form of AtGRXcp was amplified by PCR using a forward primer (5′-GGG CTC GAG AGA TCT GCG ATG GCG TCG GCT CTT ACG CCG-3′) and the AtGRXcp reverse primer. Site-directed mutagenesis was performed as described previously (35Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). A forward primer (5′-GAA TCC CGT CTC CCC ATG GCT GGA TTC TCC AAC ACT GTG GTT CAG ATT TTG-3′) and a reverse primer (CGFS; 5′-GAA TTC CGT CTC CAT GGG GAA GTC TCT CGT TCC TTT C-3′) were used for creating the C97A mutation. A forward primer (5′-GAA TCC CGT CTC CCG ATG TGT GGA GCA TCC AAC ACT GTG GTT CAG ATT TTG-3′) and the reverse primer CGFS were used for creating the F99A mutation. The fidelity of all clones was confirmed by sequencing.Yeast Strains, Expression Constructs, and Growth Assays—Saccharomyces cerevisiae wild type strain CML235 (MATa ura3-52 leu2Δ1 his3Δ200), grx5 (MATa ura3-52 leu2Δ1 his3Δ200 grx5::kanMX4) were provided by Dr. Enrique Herrero (Universitat de Lleida, Lleida, Spain) and used in all yeast experiments. Yeast Grx5 and AtGRXcp and its variants were cloned into piUGpd (36Nathan D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1409-1414Crossref PubMed Scopus (81) Google Scholar). Yeast cells were transformed by using the LiOAc method (37Ausubel F. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. ane Struhl K. Current Protocols in Molecular Biology. 2. John Wiley & Sons Inc., Hoboken, NJ1996: 13.7.1-13.7.5Google Scholar). All yeast strains were assayed on YPD medium (yeast peptone dextrose, rich medium), with or without various concentrations of H2O2, and SC medium plus six amino acids or five amino acids without lysine (21Rodríguez-Manzaneque M.T. Tamarit J. Bellí G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (390) Google Scholar, 26Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2003; 278: 6503-6509Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar).Localization of AtGRXcp-Green Fluorescent Protein (GFP) Fusions in Yeast and Plant Cells—Full-length and truncated AtGRXcp and its variants were fused to the N terminus of green fluorescent protein (GFP) using a procedure described previously (38Cheng N.-H. Liu J.-Z. Nelson R.S. Hirschi K.D. FEBS Lett. 2004; 559: 99-106Crossref PubMed Scopus (42) Google Scholar). The GFP constructs were subcloned into yeast and plant expression vectors as described previously (38Cheng N.-H. Liu J.-Z. Nelson R.S. Hirschi K.D. FEBS Lett. 2004; 559: 99-106Crossref PubMed Scopus (42) Google Scholar). The subcellular localization of the fused proteins was imaged in comparison with labeled organelle markers (chloroplasts, mitochondria, Golgi, and peroxisome) as described previously (38Cheng N.-H. Liu J.-Z. Nelson R.S. Hirschi K.D. FEBS Lett. 2004; 559: 99-106Crossref PubMed Scopus (42) Google Scholar). A peroxisome-targeted DsRed (red fluorescent protein), DsRed-per, was constructed by adding the plant peroxisome-targeting signal, KSRM, to the end of DsRed (39Trelease R.N. Lee M.S. Banjoko A. Bunkelmann J. Proto-plasma. 1995; 195: 156-167Google Scholar). The fluorescence signals were detected at 510 nm (excitation at 488 nm) for GFP, at 582 nm (excitation at 543 nm) for DsRed, and at 660 nm (excitation at 633 nm) for chlorophyll using Leica TCS SP2 AOBS confocal laser-scanning microscope. The fluorescence intensities were quantified by using the LCS software.AtGRXcp::GUS Transgenic Plants—A 397-bp DNA sequence upstream of ATG of AtGRXcp open reading frames was amplified from genomic DNA by using the following primer sets: forward primer (5′-GGC AAG CTT ATA AGT TTT AAT CGT TTA TGG GGT-3′) and reverse primer (5′-GCC TCT AGA TTT TGA CGA CTT TTA GAT TTG GAA-3′). The PCR fragment was cloned into pBI121 to replace the 35S CaMV promoter, resulting in plasmid pAtGRXcp::GUS. Agrobacterium transformation of Arabidopsis plants was performed as described previously (40Cheng N.-H. Pittman J.K. Barkla B.J. Shigaki T. Hirschi K.D. Plant Cell. 2003; 15: 347-364Crossref PubMed Scopus (179) Google Scholar). More than 50 T2 generation plants were selected for Kan resistance.Protein Oxidation Analysis—Carbonyl assays for analysis of oxidized proteins in both yeast and plant cells were performed as previously described (19Rodrígues-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (263) Google Scholar, 41Levine R.L. Williams J.A. Stadtman E.R. Shacter E. Methods Enzymol. 1994; 233: 346-357Crossref PubMed Scopus (2238) Google Scholar, 42Davletova S. Rizhsky L. Liang H. Zhong S. Oliver D.J. Coutu J. Shulaey V. Schlauch K. Mittler R. Plant Cell. 2005; 17: 268-281Crossref PubMed Scopus (732) Google Scholar). Yeast cultures of the strains (CML235 and grx5) expressing vector, Grx5, and AtGRXcp were grown in YPD media overnight at 30 °C. Half of the culture of each strain was subjected to treatment with 5 mm H2O2 for 1 h. Total proteins were extracted from cells with and without treatment. Western blot analysis was used to determine carbonyl group content. Arabidopsis chloroplasts were isolated from photosynthetic tissues of 6-week-old flowering wild type-, atgrxcp-, and AtGRXcp-overexpressing plants (43Fitzpatrick L.M. Keegstra K. Plant J. 2001; 27: 59-65Crossref PubMed Scopus (87) Google Scholar). The oxidized proteins were detected by protein gel blotting using anti-dinitrophenylhydrazone antibody (42Davletova S. Rizhsky L. Liang H. Zhong S. Oliver D.J. Coutu J. Shulaey V. Schlauch K. Mittler R. Plant Cell. 2005; 17: 268-281Crossref PubMed Scopus (732) Google Scholar) (Bethyl Laboratory, Montgomery, TX).Measurement of Iron Concentration—Yeast strains (CML235 and grx5) expressing vector, Grx5, and AtGRXcp were grown in 50 ml of selection media (-Ura) overnight at 30 °C. Each yeast culture was inoculated into 300 ml of YPD medium and grown until an A600 of 1.0 was reached. Yeast cells were harvested and washed and dried at 70 °C and subjected to inductively coupled plasma spectrometry analysis (44Havlin J.L. Soltanpour P.N. Commun. Soil Sci. Plant Anal. 1989; 14: 969-980Google Scholar). To determine the soluble iron concentration, cells were sonicated and broken with a Sonic Dismembrator (Fisher), and the intracellular iron content was examined with a QuantiChron™ iron assay kit (Bio-Assay Systems, Hayward, CA).RESULTSAtGRXcp Is a Member of the Monothiol Glutaredoxins—CXIP1 (CAX-interacting protein 1; accession number AY157988) was originally identified based on its function in a yeast assay (26Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2003; 278: 6503-6509Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar); however, we propose that CXIP1 should be reclassified as AtGRXcp. Our computational analysis revealed that AtGRXcp is similar to yeast monothiol Grxs (Grx3, -4, and -5), bacterial Grx4, PfGLP-1 from a malarial parasite, and both zebrafish and mice Grx5 (Fig. 1). This group of monothiol Grxs also contains a PICOT-HD, which is conserved in PICOTs from mammalian cells and plants (28Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Several Grxs, like yeast Grx3 and -4, and human PICOT, have an N-terminal extension; however, AtGRXcp, similar to the bacterial Grx4, and both yeast and zebrafish Grx5, does not contain the N-terminal extension (Fig. 1) (19Rodrígues-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (263) Google Scholar, 20Fernandes A.P. Fladvad M. Berndt C. Andrésen C. Lillig C.H. Neubauer P. Sunnerhagen M. Holmgren A. Vlamis-Gardikas A. J. Biol. Chem. 2005; 280: 24544-24552Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 27Wingert R.A. Galloway J.L. Barut B. Foott H. Fraenkel P. Axe J.L. Weber G.J. Dooley K. Davidson A.J. Schmid B. et al.Nature. 2005; 436: 1035-1039Crossref PubMed Scopus (320) Google Scholar). In addition, our analysis suggests that in higher plants, these monothiol Grxs exist in both monocots and dicots (Fig. 1). In Arabidopsis, there are four members of these Grxs (Fig. 1). A dithiol Grx has two cysteine residues in the active motif that are able to catalyze protein disulfides and GSH-protein mixed disulfides (11Fernandes A.P. Holmgren A. Antioxid. Redox Signal. 2004; 6: 63-74Crossref PubMed Scopus (534) Google Scholar, 17Grant C.M. Mol. Microbiol. 2001; 39: 533-541Crossref PubMed Scopus (327) Google Scholar, 18Lillig C.H. Berndt C. Vergnolle O. Lönn M.E. Hudemann C. Bill E. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8168-8173Crossref PubMed Scopus (235) Google Scholar); however, the subfamily of Grxs has only one conserved cysteine residue in the putative active motif, termed “CGFS” (Fig. 1). These observations indicate that monothiol Grxs are also conserved throughout prokaryotes and eukaryotes (Fig. 1). Based on the sequence analysis of those Grxs, we conclude that AtGRXcp is the first cloned plant monothiol Grx.AtGRXcp Is a Chloroplast-localized Monothiol Grx—Monothiol Grxs have a diverse subcellular distribution in multiple organisms. For example, yeast Grx5 is mitochondria-localized (21Rodríguez-Manzaneque M.T. Tamarit J. Bellí G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (390) Google Scholar), and Grx3 targets to nuclei (45Molina M.M. Bellí G. de la Torre M.A. Rodríguez-Manzaneque M.T. Herrero E. J. Biol. Chem. 2004; 279: 51923-51930Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), whereas a human PICOT is located in the cytosol (24Witte S. Villalba M. Bi K. Liu Y. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). In order to gain insight into the function of AtGRXcp, we fused it with GFP at its C terminus and transiently expressed this fusion protein in tobacco leaf cells. Using various organelle markers, AtGRXcp-GFP was shown to clearly target to chloroplasts rather than mitochondria, Golgi, or peroxisomes in mesophyll cells (Fig. 2A). Analysis of the AtGRXcp sequence predicts that a 63-amino acid signal peptide is present at the N terminus (data not shown). To experimentally verify this, we removed this putative signal peptide and fused the truncated AtGRXcp (AtGRXcpΔ63) with GFP for transient expression in tobacco cells. As shown in Fig. 2B, AtGRXcpΔ63-GFP no longer targeted to chloroplasts and instead was dispersed throughout the cytosol and nuclei, similar to observations with free GFP (Fig. 2B, data not shown).FIGURE 2Subcellular localization of AtGRXcp-GFP in plant cells. A, AtGRXcp-GFP is localized to chloroplasts in tobacco cells. The left panels display the transient expression of AtGRXcp-GFP in tobacco cells; central panels display fluorescence from individually labeled markers for plant organelles or fluorescing chloroplasts; and right panels show the merged images. B, a truncated AtGRXcp-GFP fusion is not targeting to chloroplasts. Scale bars, 25 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)AtGRXcp Suppresses the Sensitivity of a Yeast grx5 Mutant to H2O2—Yeast grx5 cells are growth-impaired in minimal medium and sensitive to oxidative stresses (19Rodrígues-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (263) Google Scholar, 21Rodríguez-Manzaneque M.T. Tamarit J. Bellí G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (390) Google Scholar). To examine if AtGRXcp could restore Grx5 function and suppress the sensitivity of grx5 cells to an external oxidant, H2O2, we expressed vector control, AtGRXcp, and yeast endogenous Grx5 in grx5 cells. All yeast strains grew normally in YPD liquid media (rich media) after 48 h of growth (Fig. 3A). Whereas vector-expressing grx5 cells were growth-impaired in the medium with 3 mm H2O2, both AtGRXcp- and Grx5-expressing grx5 cells grew in a similar manner to wild type cells (Fig. 3A). These observations suggest that AtGRXcp is able to suppress the sensitivity of grx5 cells to oxidative stress.FIGURE 3AtGRXcp is able to suppress the sensitivity of grx5 cells to H2O2. A and B, vector-expressing wild type cells and vector-, AtGRXcp-, AtGRXcp-GFP-, AtGRXcpΔ63-, AtGRXcpΔ63-GFP-, and Grx5-expressing grx5 cells were grown in YPD liquid media and the same media supplemented with 3.0 mm H2O2. Cell density was measured at A600 after 48 h of growth at 30 °C. Shown are two representative experiments from four independent experiments conducted. The bars indicate the S.E. (n = 8). C, subcellular localization of AtGRXcp-GFP (top) and AtGRXcpΔ63-GFP (bottom) in yeast cells. Scale bars, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Given that AtGRXcp localized to chloroplasts, which are specific to plants (Fig. 2) and yeast Grx5 is a mitochondrial Grx (21Rodríguez-Manzaneque M.T. Tamarit J. Bellí G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (390) Google Scholar), the suppression of grx5 phenotypes mandates that the subcellular localization of AtGRXcp in yeast cells differs from that seen in plants. To investigate this, AtGRXcp-GFP was expressed in yeast cells. Yeast growth assays revealed that AtGRXcp-GFP was functional and could suppress the sensitivity of grx5 cells to H2O2 (Fig. 3B). Through immunolabeling studies, AtGRXcp-GFP localized to mitochondria in yeast cells, whereas a truncated AtGRXcpΔ63-GFP was unable to target to this organelle (Fig. 3C). Targeting of AtGRXcp to mitochondria was essential for the function of this protein in yeast, since both AtGRXcpΔ63 and AtGRXcpΔ63-GFP were unable to suppress the sensitivity of grx5 cells to H2O2 (Fig. 3, A and B).AtGRXcp Is Able to Protect Cells against Protein Oxidation and Rescue the Lysine Auxotrophy of a Yeast grx5 Mutant—In grx5 cells, total protein carbonyl content is significantly increased under oxidative stress (Fig. 4A), suggesting that yeast Grx5 plays a vital role in directly protecting enzymes from oxidative damages (19Rodrígues-Manzaneque M.T. Ros J. Cabiscol E. Sorribas A. Herrero E. Mol. Cell. Biol. 1999; 19: 8180-8190Crossref PubMed Scopus (263) Google Scholar, 21Rodríguez-Manzaneque M.T. Tamarit J. Bellí G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Crossref PubMed Scopus (390) Google Scholar). In order to determine if AtGRXcp could directly reduce protein carbonylation in the grx5 cells, we performed Western blot analysi" @default.
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- W1993669834 title "AtGRXcp, an Arabidopsis Chloroplastic Glutaredoxin, Is Critical for Protection against Protein Oxidative Damage" @default.
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