Functional analysis of the putative mitochondrial copper chaperone AtCox11

Cox11 (cytochrome c oxidase 11) is an ancient and conserved protein family present in most respiring organisms. Studies of several family members, mainly in yeast and bacteria, have revealed that these proteins are in charge of Cu+ delivery to the respiratory complex IV (COX). Absence of Cox11 leads to a non-functional COX complex and a complete respiratory deficiency. Although it is assumed that homologues in other species perform the same function, experimental data supporting this notion are lacking. The aim of this work was to characterize the putative Arabidopsis homologue AtCox11 (encoded by locus At1g02410) and to determine its functions. Comparison of AtCox11 with the well-studied ScCox11 in yeast revealed that the two proteins share high similarity in their sequences (32% amino acid identity) and in the predicted secondary structures. Surprisingly, despite this high similarity AtCox11 proved not to be able to functionally replace the yeast protein in ΔSccox11 yeast deletion strains. As presumed, AtCox11 is localized to mitochondria, probably tethered to the inner mitochondrial membrane with its C-terminus facing the intermembrane space. The subsequent experimental work addressed the functions of AtCox11. To this end AtCOX11 knock-down (KD) and overexpression lines (OE) were generated and their impact on plant phenotype was investigated. KD lines that were obtained by artificial micro RNA technology, possess approximately 30% of the WT AtCOX11 mRNA levels. Overexpression resulting in 4-6 fold higher AtCOX11 mRNA levels, was achieved by placing AtCOX11 under the control of the 35S promoter. Remarkably, both KD and OE plants had reduced levels of COX complex activity (~45% and ~80%, respectively) indicating that AtCox11 is, as expected, involved in COX complex assembly. The KD and OE plants exhibited reduced root lengths and pollen germination rates (compared to WT). As both processes are dependent on respiratory energy, these phenotypic changes seemingly result from the reduced COX activity. Interestingly, the short-root phenotype in OE plants was rescued by a surplus of copper in the media, whereas copper deficiency intensified the phenotype. By contrast, KD plants did not respond to changes of the copper concentration. This difference in the copper response between KD and OE plants hints at a different cause for the reduced COX activity. It is proposed that the concentration of AtCox11 in KD plants limits the efficient insertion of Cu+ into COX, independent of the available copper concentration. In OE plants, binding of the limited copper by the high AtCox11 level may lead to a copper deficiency for the copper chaperone AtHcc1 that is required to load copper to subunit AtCoxII. Indeed, addition of copper to the media was able to rescue the phenotype. In line with these data, the analysis of the expression pattern of AtCOX11 revealed that it is expressed in tissues which require substantial mitochondrial and COX biogenesis to sustain their high metabolic and/or cell division rates. Furthermore AtCOX11 was shown to be up-regulated as part of the plant’s response to increased oxidative stress induced by the addition to the plant media of peroxides or inhibitors of respiratory complexes. The up-regulation of AtCOX11 in response to oxidative stress was corroborated with publicly available RNA microarray data and analysis of the AtCOX11 promoter, which revealed the presence of a number of potential oxidative stress responsive elements. Taken together, the experimental results presented in this thesis support the conclusion that AtCox11 is a member of the conserved Cox11 protein family. Most probably, this mitochondrial protein participates in the assembly of the COX complex by inserting Cu+ into the CuB center of the AtCoxI subunit. In addition to this expected role, the data indicate that AtCox11 might participate in cellular oxidative stress response and defense via a yet unknown mechanism