Expression of higher plant mitochondrial (mt) genes is regulated at the transcriptional, posttranscriptional, and translational levels, but the vast majority of the mtDNA and RNA-binding proteins involved remain to be identified. Plant mt single-stranded nucleic acid-binding proteins were purified by affinity chromatography, and corresponding genes have been identified. A majority of these proteins belong to a family of RNA-binding proteins characterized by the presence of an N-terminal RNA-recognition motif (RRM) sequence. They diverge in their C-terminal sequences, suggesting that they can be involved in different plant mt regulation processes. Mitochondrial localization of the proteins was confirmed both in vitro and in vivo and by immunolocalization. Binding experiments showed that several proteins have a preference for poly(U)-rich sequences. This mt protein family contains the ubiquitous RRM motif and has no known mt counterpart in non-plant species. Phylogenetic and functional analysis suggest a common ancestor with RNA-binding glycine-rich proteins (GRP), a family of developmentally regulated proteins of unknown function. As with several plant, cyanobacteria, and animal proteins that have similar structures, the expression of one of the Arabidopsis thaliana mt RNA-binding protein genes is induced by low temperatures.H igher plants require mitochondrial (mt) function for their survival, which depends on proper mtDNA maintenance and expression (1). At the structural level, the mtDNA of plants is relatively large, and in most species it is constantly reorganized by recombination between repeated sequences (2). Although large portions of mtDNA have been moved around during evolution, the plant mt genome evolves very slowly through nucleotide substitution; plant mt gene sequences have remained remarkably constant, suggesting the existence of very efficient DNA repair systems. On the other hand, the expression of plant mt genes is also complexly regulated, both at the transcriptional, posttranscriptional, and translational levels. For proper maturation of its transcripts, mitochondria puts to play complex processes of intron splicing, 5Ј and 3Ј RNA trimming, extensive RNA editing by C to U conversions, and regulation of transcript stability by secondary structures and polyadenylation (3, 4). Despite the importance of these mt processes in plant development, little is known about the factors involved, but at the core of the protein complexes must be DNA-binding proteins involved in mtDNA replication, recombination, repair and transcription, and RNA-binding proteins involved in posttranscriptional RNA maturation and translation. As a first step in the dissection of these complexes, we undertook to purify and identify nucleic acid-binding proteins from plant mitochondria. Most of the proteins identified are plant-specific, and many belong to a previously undescribed family of mt RNA-binding proteins (mRBP) characterized by the presence of an N-terminal RNA recognition motif (RRM). This family of plant mRBPs is ph...

Between the different types of Acyl-CoA dehydrogenases (ACADs), those specific for branched chain acyl-CoA derivatives are involved in the catabolism of amino acids. In mammals, isovaleryl-CoA dehydrogenase (IVD), an enzyme of the leucine catabolic pathway, is a mitochondrial protein, as other acyl-CoA dehydrogenases involved in fatty acid b-oxidation. In plants, fatty acid b-oxidation takes place mainly in peroxisomes, and the cellular location of the enzymes involved in the catabolism of branched-chain amino acids had not been definitely assigned.Here, we describe that highly purified potato mitochondria have important IVD activity. The enzyme was partially purified and cDNAs from two different genes were obtained. The partially purified enzyme has enzymatic constant values with respect to isovaleryl-CoA comparable to those of the mammalian enzyme. It is not active towards straight-chain acyl-CoA substrates tested, but significant activity was also found with isobutyryl-CoA, implying an additional role of the enzyme in the catabolism of valine. The present study confirms recent reports that in plants IVD activity resides in mitochondria and opens the way to a more detailed study of amino-acid catabolism in plant development.Keywords: plant mitochondria; isovaleryl-CoA dehydrogenase; leucine catabolism.The desaturation of the C 2 -C 3 bond of acyl-CoA esters to 2-trans-enoyl-CoA is an important step in the b-oxidation of fatty acids [1,2] and in the catabolism of branchedchain amino acids, leucine, isoleucine and valine [3,4]. In eukaryotes, this reaction can be catalyzed by two distinct type of enzymes: mitochondrial acyl-CoA dehydrogenases (ACADs), which feed reducing equivalents to the ubiquinone mitochondrial pool [5], and by peroxisomal acyl-CoA oxidases (ACOXs), which pass electrons directly on to molecular oxygen producing H 2 O 2 [2]. Both types of enzymes utilize FAD as cofactor. In mammalian tissues, b-oxidation takes place in both peroxisomes and mitochondria. In contrast, in higher plants this catabolism takes place mainly in peroxisomes [6±8], and the existence in higher plants of an additional mitochondrial b-oxidation system (and thus of ACAD enzymes) has often been considered controversial [9±11]. However, inhibition of mitochondrial b-oxidation by respiratory-chain inhibitors [9,12] strongly suggested that higher-plant mitochondria possess ACAD activities, which were identified in mitochondria from carbohydrate-starved maize root tips and sunflower embryos [12,13]. Moreover, putative plant ACAD genes could be identified on the basis of their sequence similarity with the corresponding mammalian enzymes [13]. However, a number of these putative ACADs have eventually been characterized as peroxisomal ACOXs [11,14].Among ACAD activities typically found in the mitochondria of animal cells, those specific for branched-chain substrates are involved in the catabolism of amino acids, in which the isobutyryl-CoA, 2-methyl-butyryl-CoA, and isovaleryl-CoA catabolites of Val, Ile, and Leu, respectively, und...

A method is presented for the partial purification of a plant mitochondrial active chromosome (MAC). This method is based on the presence of the mitochondrial chromosome in the insoluble mitochondrial fraction which allows for its rapid purification from the bulk of detergent-solubilized proteins by ultra-centrifugation. The resuspended MAC carrying DNA and RNA-binding proteins retains DNA synthesis and transcription activities comparable to the ones found in isolated mitochondria. In comparison, tRNA-nucleotidyl terminal transferase taken as an example of RNA modifying activities remains in the soluble fraction. MAC purification is proposed as a rapid and efficient first step in the purification of DNA-binding proteins involved in DNA replication and transcription.z 1999 Federation of European Biochemical Societies.