Identification of mitochondrial coenzyme a transporters from maize and Arabidopsis

Abstract

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.

Figure 1.

Phylogenetic analysis of known and predicted CoA transporters. A, Phylogenetic tree of the experimentally validated CoA transporters from yeast, human, and Arabidopsis, the candidate CoA transporters from Arabidopsis (At1g14560 and At4g26180) and maize (GRMZM2G420119 and GRMZM2G161299; abbreviated to ZM420119 and ZM161299), and all other Arabidopsis MCF proteins. Sequences were aligned with ClustalW; the tree was constructed by the neighbor-joining method with MEGA5. Bootstrap values (%) for 1,000 replicates are shown next to nodes; those of less than 50% are omitted. Only the tree topology is shown. The yellow highlighted sector contains the validated human and yeast mitochondrial CoA transporters and their Arabidopsis and maize homologs; the type 1 and type 2 plant carriers are indicated. The green highlighted sector contains the validated human and Arabidopsis peroxisomal CoA transporters. B, Phylogenetic distribution of type 1 and type 2 predicted CoA transporters among angiosperms, gymnosperms, and lower plants. Red boxes and blue boxes indicate the number of type 1 and type 2 sequences, respectively, represented in each genome or transcriptome. Piebald red/blue boxes indicate homologs in lower plants that could not be assigned to either type 1 or type 2.

Figure 2.

Complementation of the growth phenotype of the yeast CoA carrier deletant leu5Δ by the expression of candidate plant CoA carriers. Serial dilutions of wild-type (WT) cells harboring pYES2/CT (vector) or leu5Δ cells harboring pYES2/CT alone or encoding maize (ZM420119 and ZM161299) or Arabidopsis (At4g26180 and At1g14560) proteins were plated on YP medium containing 3% glycerol and incubated for 72 h at 30°C. pYES2/CT constructs encoding the human CoA transporter SLC25A42 or the Arabidopsis NAD⁺ transporter AtNDT1 were included as positive and negative controls, respectively.

Figure 3.

Effects of the expression of candidate plant CoA carriers on mitochondrial CoA contents of leu5Δ yeast cells. Cells were cultured in liquid YP medium containing 2% ethanol. Mitochondria were isolated from wild-type (WT) cells harboring pYES2/CT (vector) or from leu5Δ cells harboring pYES2/CT alone or encoding At1g14560, At4g26180, ZM420119, or ZM161299. pYES2/CT constructs encoding the human CoA transporter SLC25A42 or the human pyrimidine nucleotide transporter SLC25A33 were included as positive and negative controls, respectively. Data are means and se of at least three independent experiments and were subjected to one-way ANOVA followed by Bonferroni post hoc tests. Differences in CoA content between leu5Δ cells harboring pYES2/CT and other strains that are significant are indicated with asterisks: *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

Figure 4.

Protein uptake by purified pea mitochondria and chloroplasts in dual-import assays. The four CoA carrier cDNAs, plus the Arabidopsis YgfZ (AtYgfZ) and soybean alternative oxidase (GmAox) cDNAs as positive controls for chloroplast and mitochondrial import, respectively, were transcribed and translated in vitro in the presence of [³H]Leu. The translation products (TP) were incubated in the light with mixed pea chloroplasts (C) and mitochondria (M). The organelles were mock treated (−) or thermolysin treated (+) to remove adsorbed proteins and then reisolated on a Percoll gradient. Proteins were separated by SDS-PAGE and visualized by fluorography. Samples were loaded on the basis of equal chlorophyll or mitochondrial protein content next to an aliquot of the translation product. Exposure times were adjusted to give comparable band intensities in all tracks. Molecular masses are indicated on the right.

Figure 5.

Representative epifluorescence images of tobacco BY-2 cells transiently expressing maize or Arabidopsis CoA carrier candidates C-terminally fused to GFP. Cells were biolistically bombarded with plasmid DNA encoding a GFP-tagged candidate. Approximately 4 h later, cells were formaldehyde fixed, permeabilized with pectolyase and Triton X-100, immunostained for endogenous mitochondrial β-ATPase, serving as a mitochondrial marker protein, and then examined by epifluorescence microscopy. As indicated by the labels, each row of images corresponds to the fluorescence attributable to the candidate fusion protein and endogenous β-ATPase immunostaining (green and red, respectively) and the corresponding merged image. Boxes represent the portion of the cell shown at higher magnification in the panels to the right. Arrowheads indicate examples of toroidal structures containing a candidate fusion protein enclosing a punctate structure containing β-ATPase. Bar = 10 μm.

Figure 6.

Expression patterns of maize genes GRMZM2G161299 and GRMZM2G420119 (abbreviated to ZM161299 and ZM420119). Relative mRNA levels were determined by quantitative reverse transcription-PCR using the comparative cycle threshold method against three reference genes (FPGS, CUL, and UBCP). Error bars represent the se for two technical replicates of two biological replicates.