Degradation of mitochondrial alternative oxidase in the appendices of Arum maculatum.

Cyanide-resistant alternative oxidase (AOX) is a nuclear-encoded quinol oxidase located in the inner mitochondrial membrane. Although the quality control of AOX proteins is expected to have a role in elevated respiration in mitochondria, it remains unclear whether thermogenic plants possess molecular mechanisms for the mitochondrial degradation of AOX. To better understand the mechanism of AOX turnover in mitochondria, we performed a series of in organello AOX degradation assays using mitochondria from various stages of the appendices of Arum maculatum. Our analyses clearly indicated that AOX proteins at certain stages in the appendices are degraded at 30°C, which is close to the maximum appendix temperature observed during thermogenesis. Interestingly, such temperature-dependent protease activities were specifically inhibited by E-64, a cysteine protease inhibitor. Moreover, purification and subsequent nano LC-MS/MS analyses of E-64-sensitive and DCG-04-labeled active mitochondrial protease revealed an ∼30 kDa protein with an identical partial peptide sequence to the cysteine protease 1-like protein from Phoenix dactylifera. Our data collectively suggest that AOX is a potential target for temperature-dependent E-64-sensitive cysteine protease in the appendices of A. maculatum. A possible retrograde signalling cascade mediated by specific degradation of AOX proteins and its physiological significance are discussed.

Identification of a gene for pyruvate-insensitive mitochondrial alternative oxidase expressed in the thermogenic appendices in Arum maculatum.

Heat production in thermogenic plants has been attributed to a large increase in the expression of the alternative oxidase (AOX). AOX acts as an alternative terminal oxidase in the mitochondrial respiratory chain, where it reduces molecular oxygen to water. In contrast to the mitochondrial terminal oxidase, cytochrome c oxidase, AOX is nonprotonmotive and thus allows the dramatic drop in free energy between ubiquinol and oxygen to be dissipated as heat. Using reverse transcription-polymerase chain reaction-based cloning, we reveal that, although at least seven cDNAs for AOX exist (AmAOX1a, -1b, -1c, -1d, -1e, -1f, and -1g) in Arum maculatum, the organ and developmental regulation for each is distinct. In particular, the expression of AmAOX1e transcripts appears to predominate in thermogenic appendices among the seven AmAOXs. Interestingly, the amino acid sequence of AmAOX1e indicates that the ENV element found in almost all other AOX sequences, including AmAOX1a, -1b, -1c, -1d, and -1f, is substituted by QNT. The existence of a QNT motif in AmAOX1e was confirmed by nano-liquid chromatography-tandem mass spectrometry analysis of mitochondrial proteins from thermogenic appendices. Further functional analyses with mitochondria prepared using a yeast heterologous expression system demonstrated that AmAOX1e is insensitive to stimulation by pyruvate. These data suggest that a QNT type of pyruvate-insensitive AOX, AmAOX1e, plays a crucial role in stage- and organ-specific heat production in the appendices of A. maculatum.

Interaction of purified alternative oxidase from thermogenic Arum maculatum with pyruvate.

Plant alternative oxidase (AOX) activity in isolated mitochondria is regulated by carboxylic acids, but reaction and regulatory mechanisms remain unclear. We show that activity of AOX protein purified from thermogenic Arum maculatum spadices is sensitive to pyruvate and glyoxylate but not succinate. Rapid, irreversible AOX inactivation occurs in the absence of pyruvate, whether or not duroquinol oxidation has been initiated, and is insensitive to duroquinone. Our data indicate that pyruvate stabilises an active conformation of AOX, increasing the population of active protein in a manner independent of reducing substrate and product, and are thus consistent with an exclusive effect of pyruvate on the enzyme's apparent V(max).

A novel functional element in the N-terminal region of Arum concinnatum alternative oxidase is indispensable for catalytic activity of the enzyme in HeLa cells.

Alternative oxidase (AOX) is a quinol-oxygen oxidoreductase, which is known to possess a dicarboxylate diiron reaction center held in structurally postulated alpha-helical bundle. However, little is known about the structural or functional features of its N-terminal region in any organism, with the exception of a regulatory cysteine residue (CysI) in angiosperm plants. Here, we show that transcripts of two AOX1 isozymes (AcoAOX1a and AcoAOX1b) are coexpressed in thermogenic appendices of Arum concinnatum, while their enzymatic activities seem to be distinct. Namely, AcoAOX1a, an abundantly expressed transcript in vivo, shows an apparent cyanide-insensitive and n-propyl gallate-sensitive respiration during ectopic expression of the protein in HeLa cells, whereas AcoAOX1b exhibits a lower transcript expression, and appears to be totally inactive as AOX at the protein level. Our functional analyses further reveal that an E83K substitution in AcoAOX1b, which is located far upstream of CysI in the N-terminal region, is the cause of this loss of function. These results suggest the presence of a naturally occurring inactive AOX homologue in thermogenic plants. Accordingly, our results further imply that the N-terminal region of the AOX protein functionally contributes to the dynamic activities of respiratory control within the mitochondria.

[Isolation of the alternative oxidase from Arum maculatum].

There is plenty of alternate oxidase (AOX) in the inflorescences of thermogenic A rum maculatum. The isolated mitochondria exhibited a high activity, consuming oxygen on average 32 micromoles/min. The concentration of the isolated protein from mitochondria was 14.0 mg/ml. The isolated mitochondria were crashed by osmotic method to isolate matrix, membrane, and membrane soluble and insoluble proteins. The whole mitochondria, membrane and membrane soluble protein showed AOX activity while the matrix and membrane insoluble protein did not displayed AOX activity. The proteins were purified with FPLC by adding deoxycholamide dBC. The purified enzyme fraction exhibited a high specific activity, which could be kept for at least 6 months when stored at -70 degrees C. Furthermore, we used a silver stain system to identify the AOX, which showed 4 different protein bands from 30 kD to 32 kD. 2-dimensional electrophoresis showed 4 isoelectric points in the range from pH6.4 to 7.4 respectively.

Purification of the plant alternative oxidase from Arum maculatum: measurement, stability and metal requirement.

We have purified plant alternative oxidase (AOX) protein from the spadices of thermogenic Arum maculatum (cuckoo pint) to virtual homogeneity. The obtained enzyme fraction exhibits a high specific activity, consuming on average 32 micromol oxygen min(-1) mg(-1), which is completely stable for at least 6 months when the sample is stored at -70 degrees C. This exceptionally stable AOX activity is inhibited approximately 90% (I(50) approximately 10 microM) by 8-hydroxyquinoline (8-OHQ) and also, although to a lesser extent, by other metal chelators such as o-phenanthroline, alpha,alpha'-dipyridyl and EDTA. When inhibited by 8-OHQ, AOX activity is fully restored upon addition of 1.2 mM ferric iron, but neither ferrous iron nor manganese has any effect, whilst zinc decreases activity even further. Furthermore, we have developed a spectrophotometric assay to measure AOX activity in an accurate manner, which will facilitate future steady state and transient kinetic studies. The reliability of this assay is evidenced by retained stability of AOX protein during the course of the reaction, reproducibility of the measured initial rates, an observed 2:1 duroquinol-oxygen stoichiometry and by the fact that, in absolute terms, the measured rates of duroquinone formation and duroquinol disappearance are identical.