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.
Highlights
Glutaredoxins (Grxs) are ubiquitous small heat-stable disulfide oxidoreductases and members of the thioredoxin (Trx) fold protein family
AtGRXcp 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 [26]; we propose that CXIP1 should be reclassified as AtGRXcp
This group of monothiol Grxs contains a PICOT-HD, which is conserved in PICOTs from mammalian cells and plants [28]
Summary
Isolation of AtGRXcp Null Alleles—To isolate atgrxcp alleles, two T-DNA insertional mutant lines were obtained from the SALK T-DNA collection [32]. 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 (5Ј-GGG CTC GAG AGA TCT GCG ATG GCG TCG GCT CTT ACG CCG3Ј) and the AtGRXcp reverse primer. 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 (5Ј-GGC AAG CTT ATA AGT TTT AAT CGT TTA TGG GGT-3Ј) and reverse primer (5Ј-GCC TCT AGA TTT TGA CGA CTT TTA GAT TTG GAA-3Ј). Protein Oxidation Analysis—Carbonyl assays for analysis of oxidized proteins in both yeast and plant cells were performed as previously described [19, 41, 42]. 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 QuantiChronTM iron assay kit (BioAssay Systems, Hayward, CA)
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