AtCOX17, an Arabidopsis homolog of the yeast copper chaperone COX17

Abstract

We have identified a new plant gene, AtCOX17, encoding a protein that shares sequence similarity to COX17, a Cu-binding protein from yeast (Saccharomyces cerevisiae) and vertebrates that mediates the delivery of Cu to the mitochondria for the assembly of a functional cytochrome oxidase complex. The newly characterized Arabidopsis protein has six Cys residues at positions corresponding to those known to coordinate Cu binding in the yeast homolog. Moreover, we show that the Arabidopsis COX17 cDNA complements a COX17 mutant of yeast restoring the respiratory deficiency associated with that mutation. These two lines of evidence indicate that the plant protein identified here is a functional equivalent of yeast COX17 and might serve as a Cu delivery protein for the plant mitochondria. COX17 was identified by investigating the hypersensitive response-like necrotic response provoked in tobacco (Nicotiana tabacum) leaves after harpin inoculation. AtCOX17 expression was activated by high concentrations of Cu, bacterial inoculation, salicylic acid treatment, and treatments that generated NO and hydrogen peroxide. All of the conditions inducing COX17 are known to inhibit mitochondrial respiration and to produce an increase of reactive oxygen species, suggesting that gene induction occurs in response to stress situations that interfere with mitochondrial function.

Figure 1

A, Nucleotide and deduced amino acid sequence of the tobacco H26-10 cDNA. Nucleotides are numbered from the first nucleotide of the cloned cDNA. The stop codon is marked with an asterisk. B, Sequence alignment of tobacco H26-10. Sequences shown correspond to the tobacco protein, an Arabidopsis protein encoded by the entire expressed sequence tag (EST) clone 192D11T7 (GenBank accession no. AF505654), a rice (Oryza sativa) protein encoded by the EST clone C28929 (GenBank accession no. AU101012), yeast COX17 (Glerum et al., 1996), and human COX17 (Amaravadi et al., 1997). Conserved cysteinyl residues are marked by an asterisk.

Figure 2

Functional complementation of the COX17 yeast mutant. Yeast strains are as follows: 1, wild-type W303; 2, respiratory-deficient mutant W303ΔCOX17; 3, respiratory-deficient mutant W303ΔCOX17 transformed with a wild-type COX17 gene from yeast; 4, empty pYPGE15 yeast expression vector; 5, respiratory-deficient mutant (W303ΔCOX17) transformed with the AtCOX17 cDNA cloned in sense orientation into pYPGE15; and 6, respiratory-deficient mutant (W303ΔCOX17) transformed with the AtCOX17 cDNA cloned in antisense orientation into pYPGE15. Yeast strains were grown in yeast peptone dextrose Glc medium (left plate) or in yeast extract peptone dextrose glycerol containing medium (right plate). Plates were photographed at 3 d after being grown at 30°C.

Figure 3

Analysis of gene expression. RNA was extracted at different intervals from Arabidopsis leaves inoculated with the incompatible bacterium Pst DC3000 avrRpm1, the virulent strain Pst DC3000 (10⁶ colony forming units mL⁻¹), salicylic (SA), sodium nitroprusside (SNP; a generator of NO), and 3-amino-1,2,4-triazole (3AT). Inoculation with water was used as a control. Blots were hybridized with riboprobes derived from the AtCOX17 cDNA. The amount of RNA loaded was verified by ethidium bromide staining.

Figure 4

Metal-regulated expression of AtCOX17. RNA was extracted at different intervals from plants submerged in water solutions containing different concentrations of metals as indicated. RNA from water-immersed plants was used as a control. Blots were hybridized to AtCOX17 (left) and PR-1 (right). The amount of RNA loaded was verified by ethidium bromide staining.