Identification of Mitochondrial Coenzyme A Transporters from Maize and Arabidopsis

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5Δ mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.CoA acts as an acyl carrier in many reactions of primary and secondary metabolism, and some 8% of the nearly 4,900 enzymes described in the Enzyme Commission database are CoA dependent (Bairoch, 2000). CoA occupies a central position in lipid metabolism, respiration, gluconeogenesis, and other pathways (Leonardi et al., 2005). It is present in all forms of life, but while all organisms can synthesize it from pantothenate (vitamin B₅), only prokaryotes, plants, and fungi are able to synthesize pantothenate; animals obtain pantothenate from the diet (Daugherty et al., 2002; Leonardi et al., 2005; Webb and Smith, 2011).In plants, the steps that convert pantothenate to CoA are almost certainly cytosolic (Webb and Smith, 2011; Gerdes et al., 2012). CoA, however, is required in mitochondria for the citric acid cycle, in chloroplasts for fatty acid synthesis, and in peroxisomes for β-oxidation. CoA, therefore, must be imported into these organelles from the cytosol, and indeed, early work demonstrated a CoA transport system in potato (Solanum tuberosum) mitochondria (Neuburger et al., 1984). Yeast (Saccharomyces cerevisiae) and mammalian mitochondria and peroxisomes likewise import CoA because they cannot make it (Fiermonte et al., 2009; Agrimi et al., 2012b). The compartmentation of CoA in all eukaryotes appears to be closely regulated, with cytosol and organelles maintaining separate CoA pools whose levels can modulate fluxes through CoA-dependent reactions (Hunt and Alexson, 2002; Leonardi et al., 2005; De Marcos Lousa et al., 2013).Mitochondrial CoA transporters belonging to the mitochondrial carrier family (MCF) have been identified in yeast (Leu-5p; Prohl et al., 2001) and human (SLC25A42; Fiermonte et al., 2009). Furthermore, peroxisomal CoA carriers from human (SLC25A17; Agrimi et al., 2012b) and Arabidopsis (Arabidopsis thaliana; peroxisomal CoA and NAD carrier PXN]; Agrimi et al., 2012a) have also been identified. However, no transporters for CoA are known for plant mitochondria or chloroplasts (Palmieri et al., 2011; Gerdes et al., 2012).In this study, a comparative genomic analysis first identified close Arabidopsis and maize (Zea mays) homologs of the yeast and mammalian mitochondrial CoA carriers as candidates for the missing plant mitochondrial or chloroplast transporters. Experimental evidence then demonstrated that the candidate proteins transport CoA when expressed in yeast, that they are targeted to mitochondria in vitro and in planta, and that they are expressed throughout the plant.