Abstract
Alternative oxidase (AOX) and plastid terminal oxidase (PTOX) are terminal oxidases of electron transfer in mitochondria and chloroplasts, respectively. Here, taking advantage of the variegation phenotype of the Arabidopsis PTOX deficient mutant (im), we examined the functional relationship between PTOX and its five distantly related homologs (AOX1a, 1b, 1c, 1d, and AOX2). When engineered into chloroplasts, AOX1b, 1c, 1d, and AOX2 rescued the im defect, while AOX1a partially suppressed the mutant phenotype, indicating that AOXs could function as PQH2 oxidases. When the full length AOXs were overexpressed in im, only AOX1b and AOX2 rescued its variegation phenotype. In vivo fluorescence analysis of GFP-tagged AOXs and subcellular fractionation assays showed that AOX1b and AOX2 could partially enter chloroplasts while AOX1c and AOX1d were exclusively present in mitochondria. Surprisingly, the subcellular fractionation, but not the fluorescence analysis of GFP-tagged AOX1a, revealed that a small portion of AOX1a could sort into chloroplasts. We further fused and expressed the targeting peptides of AOXs with the mature form of PTOX in im individually; and found that targeting peptides of AOX1a, AOX1b, and AOX2, but not that of AOX1c or AOX1d, could direct PTOX into chloroplasts. It demonstrated that chloroplast-localized AOXs, but not mitochondria-localized AOXs, can functionally compensate for the PTOX deficiency in chloroplasts, providing a direct evidence for the functional relevance of AOX and PTOX, shedding light on the interaction between mitochondria and chloroplasts and the complex mechanisms of protein dual targeting in plant cells.
Highlights
Plant cells harbor two energy-converting organelles, mitochondria and chloroplasts, which evolved from ancient prokaryotes through different endosymbiotic events (Cavalier-Smith, 2000; Leister, 2003)
In vitro enzyme assays showed that plastid terminal oxidase (PTOX) uses PQH2 as substrate and alternative oxidase (AOX) exclusively uses UQH2 (Josse et al, 2003; Fu et al, 2012; Yu et al, 2014)
AOXs are UQH2 oxidases and PTOX is a PQH2 oxidase in vitro (Wang and Fu, 2016), AOXs could be PQH2 oxidases in planta, which was illustrated by the fact that the chloroplast-localized AOX1a and AOX2 acted as PQH2 oxidases in Arabidopsis (Fu et al, 2012)
Summary
Plant cells harbor two energy-converting organelles, mitochondria and chloroplasts, which evolved from ancient prokaryotes through different endosymbiotic events (Cavalier-Smith, 2000; Leister, 2003). Electron transfer in the cyanide-sensitive cytochrome pathway is coupled to transmembrane proton translocation and responsible for ATP synthesis (Juszczuk and Rychter, 2003). The alternative pathway, mediated by a cyanide-resistant alternative oxidase (AOX), catalyzes electron transfer from UQH2 (ubiquinol) to oxygen (Vanlerberghe and Mclntosh, 1997; Juszczuk and Rychter, 2003). This energywasteful alternative pathway oxidizes reducing equivalents without coupling to proton translocation across the mitochondrial membrane and ATP synthesis (Rogov et al, 2014; Vishwakarma et al, 2015)
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