Refined method to study the posttranslational regulation of alternative oxidases from Arabidopsis thaliana in vitro

Abstract

In isolated membranes, posttranslational regulation of quinol oxidase activities can only be determined simultaneously for all oxidases - quinol oxidases as well as cytochrome c oxidases - because of their identical localization. In this study, a refined method to determine the specific activity of a single quinol oxidase is exemplarily described for the alternative oxidase (AOX) isoform AOX1A from Arabidopsis thaliana and its corresponding mutants, using the respiratory chain of an Escherichia coli cytochrome bo and bd-I oxidase double mutant as a source to provide electrons necessary for O2 reduction via quinol oxidases. A highly sensitive and reproducible experimental set-up with prolonged linear time intervals of up to 60 s is presented, which enables the determination of constant activity rates in E. coli membrane vesicles enriched in the quinol oxidase of interest by heterologous expression, using a Clark-type oxygen electrode to continuously follow O2 consumption. For the calculation of specific quinol oxidase activity, activity rates were correlated with quantitative signal intensity determinations of AOX1A present in a membrane-bound state by immunoblot analyses, simultaneously enabling normalization of specific activities between different AOX proteins. In summary, the method presented is a powerful tool to study specific activities of individual quinol oxidases, like the different AOX isoforms, and their corresponding mutants upon modification by addition of effectors/inhibitors, and thus to characterize their individual mode of posttranslational regulation in a membranous environment.

Alternative Oxidase Isoforms Are Differentially Activated by Tricarboxylic Acid Cycle Intermediates

“…Due to the fact that AOX proteins were heterologously expressed under reducing conditions (these conditions are present in the E. coli cytosol) and that a reductant (DTT) was present during membrane vesicle isolation and activity measurements, AOX proteins are present in their reduced, and therefore activatable, state. Isoform-specific oxygen consumption was measured by linking the NADH dehydrogenases and ubiquinol pool of the E. coli respiratory chain to the heterologously expressed AOX isoform using a Clark-type oxygen electrode (Selinski et al, 2016(Selinski et al, , 2017.…”

“…Although recombinant AOX1A, AOX1C, and AOX1D from Arabidopsis are posttranslationally activated by pyruvate and glyoxylate (Rhoads et al, 1998;Umbach et al, 2002Umbach et al, , 2006Selinski et al, 2016Selinski et al, , 2017, the influence of tricarboxylic acid cycle (TCAC) intermediates on AOX activity has not been studied in detail. In soybean (Glycine max) mitochondria and submitochondrial particles, the AOX pathway also is activated by oxaloacetate (OAA) and 2-oxoglutarate (2-OG), but at higher concentrations than pyruvate Millar et al, 1996).…”

“…Besides transcriptional regulation, AOX activity has been shown to be posttranslationally regulated at the two highly conserved Cys residues (CysI and CysII) present in the N-terminal domain of the protein (Millar et al, 1993(Millar et al, , 1996Siedow, 1993, 1996;Rhoads et al, 1998;Siedow and Umbach, 2000;Umbach et al, 2006;Selinski et al, 2016Selinski et al, , 2017. Two interrelated mechanisms were identified regulating the activation/ inactivation of AOX: (1) oxidation/reduction of the disulfide bridge formed between the two CysI residues in the AOX dime; and (2) further activation of the reduced form via allosteric regulation by 2-oxo acids, also involving the conserved Cys residues (Millar et al, 1993(Millar et al, , 1996Umbach and Siedow, 1993;Rhoads et al, 1998;Umbach et al, 2006;Moore and Albury, 2008;Selinski et al, 2016Selinski et al, , 2017.…”

“…Two interrelated mechanisms were identified regulating the activation/ inactivation of AOX: (1) oxidation/reduction of the disulfide bridge formed between the two CysI residues in the AOX dime; and (2) further activation of the reduced form via allosteric regulation by 2-oxo acids, also involving the conserved Cys residues (Millar et al, 1993(Millar et al, , 1996Umbach and Siedow, 1993;Rhoads et al, 1998;Umbach et al, 2006;Moore and Albury, 2008;Selinski et al, 2016Selinski et al, , 2017.…”

“…Furthermore, these studies used mitochondria isolated from soybean cotyledons, where the only expressed AOX isoform is AOX2a (Finnegan et al, 1997), and tell us nothing about AOX1 isoforms, which are predominant in most other plants. In this work, a detailed comparison of posttranslational activation by TCAC intermediates of the three Arabidopsis isoforms AOX1A, AOX1C, and AOX1D was performed using a sensitive experimental setup with prolonged linear time intervals, based on Escherichia coli membranes enriched in individual AOX isoforms after heterologous expression, as described by Selinski et al (2016Selinski et al ( , 2017.…”