isolation-respiratory-measurements-mitochondria-from-arabidopsis

Isolation and Respiratory Measurements of Mitochondria from <em>Arabidopsis thaliana</em>

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

La Trobe University

Mitochondrial alternative oxidase (AOX) in plants is a non-proton-motive ubiquinol oxidase that is activated by redox mechanisms and 2-oxo acids. A comparative analysis of the AOX isoenzymes AOX1A, AOX1C, and AOX1D from Arabidopsis (Arabidopsis thaliana) revealed that cysteine residues, CysI and CysII, are both involved in 2-oxo acid activation, with AOX1A activity being more increased by 2-oxo acids than that of AOX1C and AOX1D. Substitution of cysteine in AOX1A by glutamate mimicked its activation by pyruvate or glyoxylate, but not in AOX1C and AOX1D. CysIII, only present in AOX1A, is not involved in activation by reduction or metabolites, but substitutions at this position affected activity. AOX1A carrying a serine residue at position CysI was activated by succinate, while correspondingly substituted variants of AOX1C and AOX1D were insensitive. Activation by glutamate at CysI and CysII is consistent with the formation of the thiohemiacetal, while succinate activation after changing CysI to serine suggests hemiacetal formation. Surprisingly, in AOX1A, replacement of CysI by alanine, which cannot form a (thio)hemiacetal, led to even higher activities, pointing to an alternative mechanism of activation. Taken together, our results demonstrate that AOX isoforms are differentially activated and that activation at CysI and CysII is additive.

Analysis of Posttranslational Activation of Alternative Oxidase Isoforms.

The cyanide-insensitive alternative oxidase (AOX) is a non-proton-pumping ubiquinol oxidase that catalyzes the reduction of oxygen to water and is post-translationally regulated by redox mechanisms and 2-oxo acids. Arabidopsis thaliana possesses five AOX isoforms (AOX1A-D and AOX2). AOX1D expression is increased in aox1a knock-out mutants from A. thaliana (especially after restriction of the cytochrome c pathway), but cannot compensate for the lack of AOX1A, suggesting a difference in regulation of these isoforms. Therefore, we analysed the different AOX isoenzymes with the aim to identify differences in their post-translational regulation. Seven tricarboxylic acid cycle intermediates (citrate, isocitrate, 2-oxoglutarate, succinate, fumarate, malate, and oxaloacetate) were tested for their influence on AOX1A, AOX1C, and AOX1D wild-type protein activity, using a refined in vitro system. AOX1C is insensitive to all seven organic acids, AOX1A and AOX1D are both activated by 2-oxoglutarate, but only AOX1A is additionally activated by oxaloacetate. Furthermore, AOX isoforms cannot be transformed to mimic one another by substituting the variable cysteine residues at position III in the protein. In summary, we show that AOX isoforms from Arabidopsis are differentially fine-regulated by tricarboxylic acid cycle metabolites (most likely depending on the N-terminal region around the highly conserved cysteine residues known to be involved in regulation by the 2-oxo acids pyruvate and glyoxylate) and propose that this is the main reason why they cannot functionally compensate for each other.

Jennifer Selinski

ARC Centre of Excellence in Plant Energy Biology,
Department of Animal,
Plant and Soil Science,
School of Life Science,
ARC Centre of Excellence in Plant Energy Biology, Department of Animal, Plant and Soil Science, School of Life Science

La Trobe University

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana