Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii

Abstract

Two cDNA clones (AOX1 and AOX2) and the corresponding genes encoding the alternative oxidases (AOXs) from Chlamydomonas reinhardtii were isolated and sequenced. The cDNAs, AOX1 and AOX2, contained open reading frames (ORFs) encoding putative proteins of 360 amino acids and 347 amino acids, respectively. For each of the ORFs, a potential mitochondrial-targeting sequence was found in the 5'-end regions. In comparison to AOX enzymes from plants and fungi, the predicted amino acid sequences of the ORFs showed their highest degree of identity with proteins from Aspergillus niger (38.1% and 37.2%) and Ajellomyces capsulatus (37% and 34.9%). Several residues supposed either to be Fe ligands or to be involved in the ubiquinol-binding site were fully conserved in both C. reinhardtii putative AOX proteins. In contrast, a cysteine residue conserved in the sequences of all higher plants and probably involved in the regulation of the enzyme activity was missing both from the AOX1 and AOX2 amino acid sequences and from protein sequences from various other microorganisms. The transcriptional expression of the AOX1 and AOX2 genes in wild-type cells and in mutant cells deficient in mitochondrial complex III activity was also investigated.

Characterization of the alternative oxidase of Chlamydomonas reinhardtii in response to oxidative stress and a shift in nitrogen source

“…Like many plant and fungi analysed to date, the C. reinhardtii AOX protein is recognized by a monoclonal antibody raised against the AOX of S. guttatum (Derzaph and Weger 1996). In tobacco (Vanlerberghe and McIntosh 1994) and Arabidopsis (Saisho et al 1997), only one gene (denoted Aox1 or Aox1a) is upregulated by stress factors, and this also seems to be the case for C. reinhardtii where, compared with wild-type cells, only Aox1 is induced in the dum15 mutant which lacks mitochondrial complex III activity (Dinant et al 2001). In Physiol.…”

“…The library, which is provided as a single high-density filter, was screened using a radiolabelled 790-bp portion of the Aox1 cDNA (Dinant et al 2001). This probe was generated using reverse transcription-polymerase chain reaction (RT-PCR) of RNA obtained from CC-2454 cells and used the primers 5 0 -CCTAGGCACGGCAGTATGAC-3 0 and 5 0 -CATTA CACT-TTTCTCCAGACA-3 0 .…”

“…In the green alga Chlamydomonas reinhardtii, two genes have been identified that encode for AOX (Dinant et al 2001), Aox1 and Aox2, with both showing the greatest similarity to the Aox gene of Aspergillis niger (Kirimura et al 1999). Similarly to the fungal enzyme, the C. reinhardtii AOX protein lacks the regulatory features, including the conserved Cys residue, found in the higher plant enzyme and thus is predicted to be a functional monomer.…”

“…Plant. 127, 2006 comparison, the other Aox genes in, for example, Arabidopsis (Saisho et al 1997), and the Aox2 gene of C. reinhardtii (Dinant et al 2001), have a transcript abundance that is constitutively low or undetectable. Interestingly, in C. reinhardtii, Aox1 was initially identified as Nar-5 one of a series of nitrate assimilation-related genes (Quesada et al 1998), which is suggestive of AOX and AP respiration having an important role in the changing metabolic and energy demands that arise with nitrate reduction (Quesada et al 2000).…”

Respiratory-deficient mutants of the unicellular green alga Chlamydomonas: A review

“…In addition, two nuclear genes encoding alternative oxidase enzymes (Aox1 and Aox2) are found, the AOX1 gene being much more transcribed than AOX2 [35].…”