Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATX1 from Arabidopsis1

Abstract

A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO 4 , CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.

Comparison of global responses to mild deficiency and excess copper levels in Arabidopsis seedlings

“…Metallochaperones responded differentially to low Cu levels in accordance to their function. For example, CCH (copper chaperone), a plantspecific chaperone, 34 was overrepresented under low vs. high Cu conditions, whereas under our experimental conditions, CCS (Cu chaperone for Cu/ZnSOD and the only chaperone for both cytosolic and chloroplastic Cu/ZnSODs) 35,36 was overrepresented under high vs. low Cu conditions (Fig. S3, ESI †).…”

Copper Transport Across Pea Thylakoid Membranes

“…CCH copper chaperones have been identified in Arabidopsis (Himelblau et al, 1998), with four genes encoding proteins predicted to be localized to plastids (Wintz and Vulpe, 2002). The experiments performed in this study were conducted in the absence of any added ATP and copper chaperones, thus making it improbable that this protein is a candidate for the Cu 21 transport activity we see in these thylakoid membrane preparations.…”

Arabidopsis NPCC6/NaKR1 Is a Phloem Mobile Metal Binding Protein Necessary for Phloem Function and Root Meristem Maintenance

“…The homolog in yeast (Saccharomyces cerevisiae) ATX1 has been well characterized (Lin and Culotta, 1995;Lin et al, 1997;Pufahl et al, 1997) and found to be involved both in Cu homeostasis by specifically delivering Cu to heavy metal P-type ATPases and in defense against oxidative stress. When overexpressed in a yeast strain lacking the superoxide dismutase gene SOD1, both At-ATX1 and At-CCH can protect the mutant from active oxygen toxicity in a Cu-dependent manner (Himelblau et al, 1998;Puig et al, 2007). In our experiments, expression of NaKR1 or the C-terminal region alone (containing the HMA domain) failed to complement the reactive oxygen toxicity phenotype of yeast sod1D mutant (see Supplemental Figure 2 online), indicating that the HMA domain of NaKR1 functions differently than At-ATX1/ At-CCH when expressed in yeast.…”

“…HMA domain proteins have been identified in the phloem sap of Brassica napus and Cucurbita maxima (Giavalisco et al, 2006;Lin et al, 2009). In Arabidopsis, HMA domain protein CCH is found in the phloem and is thought to transport Cu out of senescing tissues (Himelblau et al, 1998;Mira et al, 2001). nakr1-1 seedling growth (for 2 weeks) was not affected more than the wild type when Zn 2+ (up to 15 mM) or Cu 2+ (up to 25 mM) was included in the growth medium (data not shown).…”