Eukaryotic cells contain a population of mitochondria, variable in number and shape, which in turn contain multiple copies of a tiny compact genome (mtDNA) whose expression and function is strictly coordinated with the nuclear one. mtDNA copy number varies between different cell or tissues types, both in response to overall metabolic and bioenergetics demands and as a consequence or cause of specific pathological conditions. Here we present a novel and reliable methodology to assess the effective mtDNA copy number per diploid genome by investigating off-target reads obtained by whole-exome sequencing (WES) experiments. We also investigate whether and how mtDNA copy number correlates with mitochondrial mass, respiratory activity and expression levels. Analyzing six different tissues from three age- and sex-matched human individuals, we found a highly significant linear correlation between mtDNA copy number estimated by qPCR and the frequency of mtDNA off target WES reads. Furthermore, mtDNA copy number showed highly significant correlation with mitochondrial gene expression levels as measured by RNA-Seq as well as with mitochondrial mass and respiratory activity. Our methodology makes thus feasible, at a large scale, the investigation of mtDNA copy number in diverse cell-types, tissues and pathological conditions or in response to specific treatments.
Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin K m and V max values were 4.4^1.3 mm and 35^5 pmol´min 21´( mg protein) 21 , respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent K m and apparent V max values for FMN were 5.4^0.9 mm and 22.9^1.4 pmol´min 21´( mg matrix protein) 21 , respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.Keywords: FAD synthetase; flavin cofactors; mitochondria; riboflavin; transport.Although the biochemistry of mammalian mitochondria has been investigated in depth, the mechanisms of uptake and/or processing of vitamins and/or vitamin-derived cofactors, often necessary for assembly with their apoenzymes already imported from the cytosol, remain to be fully elucidated.The uptake of certain vitamins by mitochondria has already been reported to occur via different mechanisms, including diffusion followed by binding to intramitochondrial protein(s) [1], simple diffusion [2±4] and carrier-mediated transport [5,6]. Mitochondria are able to synthesize certain cofactors from taken up precursors, namely adenosylcobalamin [7] and CoA [8]. In contrast, mitochondria can take up cytosolically synthesized cofactors. Diffusion followed by binding to a specific high-affinity intramitochondrial protein has been proposed for pyridoxal 5 H -phosphate uptake [2], whereas thiamine diphosphate (TPP) and CoA can enter the mitochondria via a carrier-mediated process [9±11]. NAD synthesis from externally added nicotinamide mononucleotide has been reported in rat liver mitochondria (RLM) [12], while NAD permeation in mitochondria has also been shown in human cells [13].The above-described state of the art requires investigation regarding the existence of mitochondrial translocators and enzymes involved in vitamin and/or vitamin cofactor transport and metabolism in mitochondria. In particular, because RLM contain a huge amount of FAD and FMN, elucidation of the processes by which mitochondria provide and re...
In order to gain some insight into mitochondrial flavin biochemistry, rat liver mitochondria essentially free of lysosomal and microsomal contamination were prepared and their capability to metabolise externally added and endogenous FAD and FMN tested both spectroscopically and via HPLC.The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD+FMN and FMN-riboflavin conversion, respectively, is shown. They differ from each other and from extramitochondrial enzymes, as judged by their pH profile and inhibitor sensitivity, and can be separated in a partial FAD pyrophosphatase purification.Digitonin titration and subfractionation experiments show that FAD pyrophosphatase is located in the outer mitochondrial membrane and FMN phosphohydrolase in the intermembrane space. Since these enzymes can metabolise endogenous FAD and FMN, which are made available by using both Triton X-100 and the effector oxaloacetate, a proposal is made that FAD pyrophosphatase and FMN phosphohydrolase play a major role in mitochondrial flavoprotein turnover.
Evidence is given that mitochondria isolated from Saccharomyces cerevisiae can take up externally added riboflavin and synthesise from it both flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) probably due to the existence of the mitochondrial riboflavin kinase already reported and the novel mitochondria FAD synthetase. Moreover Saccharomyces cerevisiae mitochondria can export the newly synthesised flavin derivatives to the extramitochondrial phase. This has been proven to take place with 1:1 stoichiometry with riboflavin decrease outside mitochondria, thus showing that flavin traffic occurs across the mitochondrial membranes.z 1998 Federation of European Biochemical Societies.
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