Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt ^– parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COXI have been isolated. The possibility that the inactivation of the other mitochondrial genes is lethal for the cells is discussed.     

Kinetic mechanism of the exchanges catalysed by the adenine-nucleotide carrier

Initial rates of the exchange ADPin/ADPout catalysed by the adenine-nucleotide carrier of rat-heart mitochondria have been studied under conditions where internal and external ADP may be varied. The initial rate was measured within 1 s by the carboxyatractyloside-stop method, using a rapid-mixing technique. The double-reciprocal plots v0(-1) versus [ADP]out-1 at different internal-ADP concentrations and v0(-1) versus [ADP]in-1 at different external-ADP concentrations exhibit straight-line relationships having a common point of intersection on the axis of ordinates. These results demonstrate the essential role of a ternary complex and thus exclude the ping-pong mechanism generally accepted. The kinetic equation implies a strong positive cooperativity in the binding of the two substrates. Two models are proposed: (a) the ternary complex performs the exchange and the transport of the substrates in a single step; (b) the carrier is mobile and transports the substrates one by one, the formation of a ternary complex being needed to release the first product.     

Mitochondrial Respiratory Chain Complex Patterns from Acanthamoeba castellanii and Lycopersicon esculentum: Comparative Analysis by BN-PAGE and Evidence of Protein–Protein Interaction Between Alternative Oxidase and Complex III

We have previously shown that a kinetic interplay exists between the cytochrome pathway and the alternative oxidase in mitochondria from amoeba Acanthamoeba castellanii . Native interaction analyses using blue native gel electrophoresis coupled to denaturating electrophoresis and immunodetection have indicated associations between alternative oxidase and oxidative phosphorylation complexes in both amoeba and tomato mitochondria. These associations are dependent on the expression level of alternative oxidase according to the physiological state in both organisms. Alternative oxidase associates broadly with large complexes of the respiratory chain when it is expressed in large amount, i.e., in ripe tomato and exponentially growing amoeba. On the contrary, alternative oxidase interacts specifically with complex III even if expression of the oxidase is low, i.e., in green tomato and stationary phase amoeba. This specific interaction represents a higher level of regulation driven by protein-protein interactions leading to a direct kinetic interplay between the cytochrome pathway and alternative oxidase in both plant and amoeba mitochondria.     

Kinetics of Light Emission and Oxygen Consumption by Bioluminescent Bacteria

Oxygen plays a key role in bacterial bioluminescence. The simultaneous and continuous kinetics of oxygen consumption and light emission during a complete exhaustion of the exogenous oxygen present in a closed system has been investigated. The kinetics are performed with Vibrio fischeri, V. harveyi, and Photobacterium phosphoreum incubated on respiratory substrates chosen for their different reducing power. The general patterns of the luminescence time courses are different among species but not among substrates. During steady-state conditions, substrates, which are less reduced than glycerol, have, paradoxally, a better luminescence efficiency. Oxygen consumption by luciferase has been evaluated to be 17% of the total respiration. Luciferase is a regulatory enzyme presenting a positive cooperative effect with oxygen and its affinity for this final electron acceptor is about 4–5 times higher than the one of cytochrome oxidase. The apparent Michaelis constant for luciferase has been evaluated to be in the range of 20 to 65 nM O₂. When O₂ concentrations are as low as 10 nM, luminescence can still be detected; this means that above this concentration, strict anaerobiosis does not exist. By n-butyl malonate titration, it was clearly shown that electrons enter the luciferase pathway only when the cytochrome pathway is saturated. It is suggested that, in bioluminescent bacteria, luciferase acts as a free-energy dissipating valve when anabolic processes (biomass production) are impaired.     

The energy-conserving and energy-dissipating processes in mitochondria isolated from wild type and nonripening tomato fruits during development on the plant

Bioenergetics of tomato (Lycopersicon esculentum) development on the plant was followed from the early growing stage to senescence in wild type (climacteric) and nonripening mutant (nor, nonclimacteric) fruits. Fruit development was expressed in terms of evolution of chlorophyll a content allowing the assessment of a continuous time-course in both cultivars. Measured parameters: the cytochrome pathway-dependent respiration, i.e., the ATP synthesis-sustained respiration (energy-conserving), the uncoupling protein (UCP) activity-sustained respiration (energy-dissipating), the alternative oxidase(AOX)-mediated respiration (energy-dissipating), as well as the protein expression of UCP and AOX, and free fatty acid content exhibited different evolution patterns in the wild type and nor mutant that can be attributed to their climacteric/nonclimacteric properties, respectively. In the wild type, the climacteric respiratory burst observed in vitro depended totally on an increse in the cytochrome pathway activity sustained by ATP synthesis, while the second respiratory rise during the ripening stage was linked to a strong increase in AOX activity accompanied by an overexpression of AOX protein. In wild type mitochondria, the 10-M linoleic acid-stimulated UCP-activity-dependent respiration remained constant during the whole fruit development except in senescence where general respiratory decay was observed.     

Fatty acid efficiency profile in uncoupling of <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> mitochondria

A profile of free fatty acid (FFA) specificity in Acanthamoeba castellanii mitochondrial uncoupling is described. The FFA uncoupling specificity was observed as different abilities to stimulate resting respiration, to decrease resting membrane potential, and to decrease oxidative phosphorylation efficiency. Tested unsaturated FFA (C18–20) were more effective as uncouplers and protonophores when compared to tested saturated FFA (C8–18), with palmitic acid (C16:0) as the most active. As FFA efficiency in mitochondrial uncoupling is related to physiological changes of fatty acid composition (and thereby FFA availability) during growth of amoeba cells, it could be a way to regulate the activity of an uncoupling protein and thereby the efficiency of oxidative phosphorylation during a cell life of this unicellular organism. Aleksandra Swida and Małgorzata Czarna contributed equally to this work.     

Mitoproteome plasticity of rat brown adipocytes in response to cold acclimation

Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein patterns found in rat control and cold-acclimated BAT was performed. A total of 58 proteins exhibiting significant differences in their abundance was unambiguously identified. Proteins implicated in the major catabolic pathways were up-regulated as were ATP synthase and mitofilin. Moreover, these results support the fact that adipocytes can balance their ATP synthesis and their heat production linked to UCP1-sustained uncoupling.     

Steatosis-induced proteomic changes in liver mitochondria evidenced by two-dimensional differential in-gel electrophoresis

Steatosis encompasses the accumulation of droplets of fats into hepatocytes. In this work, we performed a comparative analysis of mitochondrial protein patterns found in wild-type and steatosis-affected liver using the novel technique two-dimensional differential in-gel electrophoresis (2D-DIGE). A total of 56 proteins exhibiting significant difference in their abundances were unambiguously identified. Interestingly, major proteins that regulate generation and consumption of the acetyl-CoA pool were dramatically changed during steatosis. Many proteins involved in the response to oxidative stress were also affected.