Fig. 1Identification of a novel variant of mitochondrial trans-2-enoyl-CoA reductase (MECR; cytosolic form of MECR [cMECR]) cDNA. (A) The genomic structure of MECR and cMECR and their coding regions are shown. Inverted triangles indicate the start and stop codons. The dotted-line box indicates the inserted nucleotide sequence. (B) Alignment of the MECR and cMECR sequences. The positions of the upper and lower primers are marked with arrows. (C) Expression patterns of MECR and cMECR in various tissues. The upper polymerase chain reaction band is cMECR, and the lower band is MECR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a positive control. Br, brain; He, heart; Ki, kidney; Li, liver; Lu, lung; Pa, pancreas; Sp, spleen; Th, thymus. aEach start codon.
Fig. 2Subcellular localization of mitochondrial trans-2-enoyl-CoA reductase (MECR) and cytosolic form of MERC (cMECR) in HeLa cells. (A) The two constructs used in this study: MECR and cMECR were tagged at the C-terminus with enhanced green fluorescent protein (EGFP; green oval). The black box in MECR indicates the mitochondrial targeting domain. HeLa cells were transiently transfected with (B) MECR-EGFP or (C) cMECR-EGFP and then stained with Mito Tracker, a mitochondrial marker (red). Scale bar=20 µm.
Fig. 3Cytosolic form of mitochondrial trans-2-enoyl-CoA reductase (cMECR) directly interacts with peroxisome proliferator-activated receptor α (PPARα) in vitro. (A) Yeast two-hybrid assay between cMECR and PPARα: on a Trp-/Leu-/His- plate, colonies indicate an interaction between the two genes. (B) Coimmunoprecipitation assays with cMECR and PPARα: cMECR-FLAG was cotransfected with hemagglutinin (HA)-PPARα in HeLa cells. The lysates were immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-HA antibody. (C) Colocalization of cMECR and PPARα: HeLa cells were transiently transfected with cMECR-enhanced green fluorescent protein (EGFP; green) and mCherry-PPARα (red). Scale bar=20 µm.
Fig. 4Cytosolic form of mitochondrial trans-2-enoyl-CoA reductase (cMECR) potentiates peroxisome proliferator-activated receptor α (PPARα) activity. (A) Schematic diagram to explain the use of the venus fusion proteins for bimolecular fluorescence complementation (BiFC). (B) BiFC of cMECR with PPARα: plasmids containing cMECR-N terminal fragment of venus (VN) and C terminal fragment of venus (VC)-PPARα were cotransfected into HeLa cells. These cells were stained with 4',6-diamidino-2-phenylindole (DAPI). Venus fluorescence is pseudocolored green. Scale bar=20 mm. (C) Luciferase assay of PPARα: PPARα activity was measured in HeLa cells expressing the PPAR-Luciferase reporter plasmid, HA-PPARα, and cMECR-FLAG. All values are mean±s.e.m. P values were obtained with Student's t test. aP<0.01.
Supplementary Fig. 1(A) Nucleotide sequence showing exon/intron junctions for the generation of cytosolic form of mitochondrial trans-2-enoyl-CoA reductase (cMECR): all exon-intron boundaries conform to consensus splice-donor and splice-acceptor sites (GT-AG). (B) Amino acid alignment of rat cMECR with human MECR isoform 2: the amino acid sequences of both proteins were aligned using the Uniprot alignment program. Sequence identity is 87.2%. Asterisks mean same amino acid.
Supplementary Fig. 2(A) Schematic drawing of the effect of enhanced green fluorescent protein (EGFP) tags on the localization of mitochondrial trans-2-enoyl-CoA reductase (MECR). (B) N-terminal EGFP-tagged MECR is mostly primarily located in cytosolic regions. C-terminal EGFP-tagged MECR localizes to mitochondrial regions. HeLa cells were transiently transfected with EGFP-MECR or MECR-EGFP. Scale bar=20 µm.
Supplementary Fig. 3Localization of mitochondrial trans-2-enoyl-CoA reductase (MECR) and peroxisome proliferator-activated receptor α (PPARα): wild-type MECR is localized in cytosolic regions, whereas PPARα is localized in nuclear regions. HeLa cells were transiently transfected with MECR-enhanced green fluorescent protein (EGFP; green) and mCherry-PPARα (red). Scale bar=20 µm.