CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.
Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. During isolation of a COT clone from a rat liver library, a cDNA in which exon 2 was repeated, was characterized. Reverse transcription-PCR amplifications of total RNAs from rat liver showed a three-band pattern. Sequencing of the fragments revealed that, in addition to the canonical exon organization, previously reported [Choi, S. J. et al. (1995) Biochim. Biophys. Acta 1264, 215-222], there were two other forms in which exon 2 or exons 2 and 3 were repeated. The possibility of this exonic repetition in the COT gene was ruled out by genomic Southern blot. To study the gene expression, we analyzed RNA transcripts by Northern blot after RNase H digestion of total RNA. Three different transcripts were observed. Splicing experiments also were carried out in vitro with different constructs that contain exon 2 plus the 5 or the 3 adjacent intron sequences. Our results indicate that accurate joining of two exons 2 occurs by a trans-splicing mechanism, confirming the potential of these structures for this process in nature. The trans-splicing can be explained by the presence of three exon-enhancer sequences in exon 2. Analysis by Western blot of the COT proteins by using specific antibodies showed that two proteins corresponding to the expected M r are present in rat peroxisomes. This is the first time that a natural trans-splicing reaction has been demonstrated in mammalian cells.In trans-splicing, two pre-mRNAs are processed to produce a mature transcript that contains exons from both precursors. This process is believed to proceed through two trans-esterification steps that result in the linking of the two exons by a normal 3Ј-5Ј phosphodiester bond. This process has been described mostly in trypanosoma, nematodes, plant͞algal chloroplasts, and plant mitochondria (1).Trans-splicing of artificial pre-mRNAs in mammalian cells in vitro has been reported but with some limitations (2-5). In addition, spliced leader RNAs from nematodes or from Simian virus 40 can be accurately trans-spliced in transfected COS cells, which reveals functional conservation in the splicing machinery between lower eukaryotes and mammals and demonstrated the potential for trans-splicing in mammalian cells (6). Studies in vitro also have shown that a synthetic pre-mRNA substrate containing an exon and a 5Ј donor splice site can be efficiently trans-spliced to another synthetic pre-mRNA (3Ј trans-splicing substrate) if this contains either exonic enhancers or a downstream 5Ј splice site (7-8). Several examples of possible natural trans-splicing in mammalian cells have been reported (9-12), but none of these trans-splicing have been demonstrated in vitro.During our current investigation on the carnitine octanoyltransferase (COT) gene, that encodes for an enzyme, which transports medium-chain fatty acids through the peroxisome, we isolated a cDNA COT clone, which had exon 2 repeated. We report here that the pre-mRNA of COT from rat liver produces...
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