Functional and conformational properties of the exclusive C-domain from the Arabidopsis copper chaperone (CCH).

The Arabidopsis thaliana copper chaperone (CCH) is a small copper binding protein involved in copper trafficking. When compared to homologues from other eukaryotes, CCH has two different domains; the conserved N-domain and the plant-exclusive C-domain, a C-terminal extension with an unusual amino-acid composition. In order to characterize this extra C-domain, the CCH protein, the N-domain and the C-domain were all expressed separately in heterologous systems. While the N-domain retained the copper chaperone and antioxidant properties described for the yeast Atx1 and human HAH1 counterparts, the C-domain displayed particular structural properties that would be necessary to optimize copper homoeostasis in plant cells where it could be responsible for the metallochaperone plant-exclusive intercellular transport. The whole CCH protein and the C-domain, but not the N-domain, displayed altered SDS/PAGE mobilities. CD spectroscopy showed that the N-domain fold is representative of an alpha/beta protein, while the C-domain adopts an extended conformation.

Evidence for the plant-specific intercellular transport of the Arabidopsis copper chaperone CCH.

Arabidopsis copper chaperone (CCH) belongs to a family of eukaryotic proteins that participates in intracellular copper homeostasis by delivering this metal to the secretory pathway. In this work we show that the CCH protein is mainly located along the vascular bundles of senescing leaves and petioles, as shown by tissue prints and immunohistochemical detection. CCH protein also accumulates in stem sieve elements and is collected in phloem exudates. Accordingly, Arabidopsis CCH is the only member of the metallochaperone family described to function intercellularly to date. Moreover, the CCH protein remains stable when plants are subjected to excess copper that causes a rapid and specific decrease in its mRNA. These facts point to a role for CCH in copper mobilization from decaying organs towards reproductive structures, as a result of metalloprotein breakdown.

Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis.

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 CuSO4, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.

An Antisense Gene Stimulates Ethylene Hormone Production during Tomato Fruit Ripening.

The ripening of many fruits is controlled by an increase in ethylene hormone concentration. E8 is a fruit ripening protein that is related to the enzyme that catalyzes the last step in the ethylene biosynthesis pathway, 1-aminocyclopropane-1-carboxylic (ACC) oxidase. To determine the function of E8, we have transformed tomato plants with an E8 antisense gene. We show here that the antisense gene inhibits the accumulation of E8 protein during ripening. Whereas others have shown that reduction of ACC oxidase results in reduced levels of ethylene biosynthesis, we find that reduction of the related E8 protein produces the opposite effect, an increase in ethylene evolution specifically during the ripening of detached fruit. Thus, E8 has a negative effect on ethylene production in fruit.

Protection and enhancement of ribulose 1,5 bisphosphate carboxylase activity by exogenous proteins.

When assayed in vitro, the activity of the photosynthetic enzyme ribulose 1,5 bisphosphate carboxylase oxygenase is both enhanced and protected from spontaneous decay by exogenous proteins such as hemoglobin, serum albumin, and aldolase. Other proteins and amino acids tested are either ineffective (lysozyme, ferritin, lysine, and cysteine) or afford only partial protection (catalase, glycine, and phenylalanine). Protective proteins do not bind to, or exchange disulfides with, ribulose 1.5 bisphosphate carboxylase/oxygenase. Since their effect can be mimicked by reductively treated detergents such as Triton X-100, it appears that proteins protect from decay by quenching the spontaneous oxidative degradation and inhibiting surface adsorption which could lead to enzyme unfolding. Release of adsorbed molecules from the container surface is likely to be the cause of carboxylase activity enhancement.

Increased susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolytic degradation caused by oxidative treatments.

The susceptibility of the chloroplastic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase to proteolysis by trypsin, chymotrypsin, proteinase K, and papain is enhanced by oxidative treatments including spontaneous oxidation of cysteines. Proteinases exhibit a high specificity for the oxidized inactive form of the carboxylase, cleaving its large subunit. Treatment of the inactive enzyme with dithiothreitol results in partial recovery of both carboxylase activity and resistance to proteolysis. This behavior may explain the specific degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase that occurs in vivo during leaf senescence.