An uncoupling protein (UCP) has been identified in mitochondria from؉ cycling, early during phylogenesis before the major radiation of phenotypic diversity in eukaryotes and could occur in the whole eukaryotic world.

Mitochondria of the protozoa Acanthamoeba castellanii possess a cyanide-insensitive oxidase cross-reacting with monoclonal antibodies raised against the plant alternative oxidase. Immunoblotting revealed three monomeric forms (38, 35, and 32 kDa) and very low amounts of a single 65 kDa dimeric form. Cross-linking studies suggest that while in plants the alternative oxidase occurs as a dimer, in amoeba it functions as a monomer. Immunologically detectable protein levels change with the age of amoeba cell culture. Increased amounts of the 35 kDa protein are accompanied by an increase in the activity of cyanideresistant respiration.

Amoeba mitochondria possess a respiratory chain with two quinol-oxidizing pathways: the cytochrome pathway and the cyanide-resistant alternative oxidase pathway. The ADP/O method, based on the non-phosphorylating property of alternative oxidase, was used to determine contributions of both pathways in overall state 3 respiration in the presence of GMP (an activator of the alternative oxidase in amoeba) and succinate as oxidizable substrate. This method involves pair measurements of ADP/O ratios plus and minus benzohydroxamate (an inhibitor of the alternative oxidase). The requirements of the method are listed and verified. When overall state 3 respiration was decreased by increasing concentrations of n-butyl malonate (a non-penetrating inhibitor of succinate uptake), the quinone reduction level declined. At the same time, the alternative pathway contribution decreased sharply and became negligible when quinone redox state was lower than 50%, whereas the cytochrome pathway contribution first increased and then passed through a maximum at a quinone redox state of 58% and sharply decreased at a lower level of quinone reduction. This study is the first attempt to examine the steady-state kinetics of the two quinol-oxidizing pathways when both are active and to describe electron partitioning between them when the steady-state rate of the quinone-reducing pathway is varied.The mitochondrial respiratory chain of the amoeba Acanthamoeba castellanii possesses, like that of plant mitochondria (1), the following: (i) a cyanide-and antimycin-resistant alternative oxidase (AOX) 1 in addition to the conventional cytochrome c oxidase (2, 3); (ii) the rotenone-insensitive external NADH dehydrogenase, located on the outer surface of the inner mitochondrial membrane (4, 5); and (iii) two internal NADH dehydrogenases, the rotenone-sensitive complex I and the non-electrogenic rotenone-insensitive dehydrogenase (6).As in mitochondria from most higher plants, many fungi, and protozoa (7), the alternative pathway of amoeba mitochondria branches from the main respiratory chain at the level of quinone (Q), and electron flux through AOX is not coupled to the generation of protonmotive force and ADP phosphorylation (2). The cyanide-resistant respiration of amoeba mitochondria is strongly stimulated by purine nucleoside 5Ј-monophosphates: AMP, GMP (with the highest efficiency), and IMP (2,8,9). A similar effect of purine mononucleotides on the activity of the alternative pathway was observed in other microorganisms: Euglena gracilis (10), Moniliella tomentosa (11), Neurospora crassa (12), Paramecium tetraurelia (13), and Hansenula anomala (14). In contrast, the alternative oxidase of higher plant mitochondria is stimulated by ␣-keto acids, like pyruvate (7, 15, 16), but not by purine mononucleotides, whereas the ␣-keto acids do not stimulate the alternative respiration in amoeba mitochondria (17). These properties emphasize an important difference at the level of allosteric regulation between both types of AOX. However, monoclonal antibodie...

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Mitochondria of amoeba Acanthamoeba castellanii in addition to the conventional cytochrome pathway possess, like plant mitochondria, a cyanide-resistant alternative quinol oxidase. In mitochondria isolated from amoeba batch culture grown temporarily at low temperature (6 degrees C), higher respiration was accompanied by lower coupling parameters as compared to control culture (grown at 28 degrees C). In the presence of benzohydroxamate, respiratory rates and coupling parameters were similar in both types of mitochondria indicating that growth in cold conditions did not disturb the cytochrome pathway. Increased contribution of alternative oxidase in total mitochondrial respiration in low-temperature-grown amoeba cells was confirmed by calculation of its contribution using ADP/O measurements. Furthermore, in mitochondria from low-temperature- grown cells the content of the alternative oxidase was increased and correlated with the increase in the unstimulated and GMP-stimulated cyanide-resistant respiratory activity. A possible physiological role of higher activity of alternative oxidase as response to growth at a low temperature in unicellular organisms, such as amoeba, is discussed.

It has been shown recently that in Saccharomyces cerevisiae mitochondria depleted of the VDAC channel (delta por1 mitochondria), the TOM complex channel substitutes for the VDAC channel. The additional function of the TOM complex channel is probably facilitated by the upregulation of nuclear-encoded components of the TOM complex as has been shown for Tom40 (a major component of the channel) and Tom70 (one of the surface receptors). Here we report that in S. cerevisiae cells the VDAC channel seems to be an important signal in the expression of the TOM complex components. S. cerevisiae cells depleted of the VDAC channel (delta por1 cells) contain distinctly increased levels of Tom40mRNA, and Tom70mRNA, but their synthesis and translation are affected differentially by the applied inhibitors of transcription and translation. Consequently, it may be concluded that depletion of the VDAC channel might influence differentially the expression of TOM40 and TOM70 genes.

Mitochondria of Acanthamoeba castellanii possess a cyanide-resistant GMP-stimulated ubiquinol alternative oxidase in addition to the cytochrome pathway. In a previous work it has been observed that an interaction between the two ubiquinol-oxidizing pathways exists in intact A. castellanii mitochondria and that this interaction may be due to a high sensitivity of the alternative oxidase to matrix pH. In this study we have shown that the alternative oxidase activity reveals a pH-dependence with a pH optimum at 6.8 whatever the reducing substrate may be. The GMP stimulation of alternative oxidase is also strongly dependent on pH implicating probably protonation/deprotonation processes at the level of ligand and protein with an optimum pH at 6.8. The ubiquinone redox state-dependence of alternative oxidase activity is modified by pH in such a way that the highest activity for a given ubiquinone redox state is observed at pH 6.8. Thus pH, binding of GMP, and redox state of ubiquinone collaborate to set the activity of the GMP-stimulated alternative oxidase in isolated A. castellanii mitochondria. The high pH sensitivity of the alternative oxidase could link inactivation of the cytochrome pathway proton pumps to activation of the alternative oxidase with acceleration of redox free energy dissipation as a consequence.

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