Evidnece that rat liver mitochondrial and cytosolic fumarases are synthesized from one species of mRNA by alternative translational initiation at two in‐phase AUG codons

Abstract

Rat liver contains two isozymes of fumarase, mitochondrial and cytosolic enzymes. Recently, we suggested that the precursors of both isozymes might be synthesized by one species of mRNA [Suzuki, T., Sato, M., J . Biol. Chem. 264, 2581-25861. To examine this possibility, we have isolated and characterized rat genomic clones for fumarase. The isolated clones covered almost all of the 5' half of the fumarase gene consisting of five exons. The first exon contained the whole 5' non-coding region and the signal peptide of mitochondrial precursor. The second exon encoded 45 amino acid residues of both mature proteins, starting from the N-terminal alanine. By using the boundary region of the first intron and the second exon as an S1-nuclease-analysis probe, we obtained conclusive evidence that rat liver contains no other mRNA specific for the cytosolic isozyme of fumarase. Two transcription-initiation sites were identified by further S1-nuclease-mapping analysis and were shown to be located very close to each other, differing by only four bases in length. Therefore, these sites were considered to be functionally the same. The results obtained by hybridselected translation, with a DNA fragment of the 5' non-coding region as a hybridization probe for selecting mRNA, were consistent with the above findings. We found a plausible secondary structure within the 5' non-coding mRNA sequence that may impede initiation and so alter the efficiency of translation. We also discuss the mechanism regulating translational initiation.We have reported that rat liver contains two isozymes of fumarase, present in almost equal amounts in the mitochondrial and cytosolic fractions [ l~ 41. The mitochondrial and cytosolic isozymes are synthesized with and without a presequence peptide at the N-terminus, respectively [5], but the primary structure of the two mature enzymes is the same [6, 71, except that the N-terminal amino acid of the cytosolic isozyme is acetylated [4].Recently, we isolated a cDNA clone (pRFum9) for rat liver fumarase containing the whole coding region of the precursors of both the mitochondrial and cytosolic isozymes, but only 25 nucleotides of the 5' non-coding region [6]. This clone contains two in-phase AUG codons separated by a nucleotide segment encoding the signal peptide of the mitochondrial precursor. This characteristic structure of fumarase mRNA together with experimental results obtained by hybridselected translation and primer-extension studies suggested that the two isozymes are translated from a single mRNA by alternative use of the two in-phase translation-initiation sites

The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases.

“…A different type of regulation has been described for the yeast genes encoding isopentenyl pyrophosphate:tRNA isopentenyltransferase (MODS) and ATP(CTP):tRNA nucleotidyltransferase (CCA1) and the rat fumarase gene (Suzuki et al, 1992). In the case of MODS, two transcripts are synthesized, and both contain two in-frame AUG codons in the 5' region.…”

“…The CCA7 gene and the rat fumarase genes also have heterogenous transcription start sites leading to mRNAs carrying two or three in-frame 5'AUG codons (Suzuki et al, 1992;Wolfe et al, 1994). For both genes, the most active AUG in terms of translation initiation appears to be the downstream one, despite the presente of one or two upstream AUG codons.…”

A Single Precursor Protein for Ferrochelatase-I fromArabidopsis Is Imported in Vitro into Both Chloroplasts and Mitochondria

“…Furthermore, although until recently it was assumed that multiple isoforms of an enzyme located in different compartments were encoded by separate nuclear genes, as is the case for superoxide dismutase (33) and malate dehydrogenase (34), examples of proteins targeted to more than one location are becoming more common. For example, the mitochondrial and cytosolic isoforms of rat fumarase (35) and isopentenyl-pyrophosphate:tRNA isopentenyltransferase and ATP(CTP):tRNA nucleotidyltransferase from yeast (36) are each the products of a single gene, as are the cytosolic and mitochondrial forms of alanine aminoacyl-tRNA synthetase in Arabidopsis (37). Analysis of transcription start sites in the latter gene showed the presence of two potential AUG codons in some transcripts, suggesting the possibility that the same message could give rise to two different polypeptides.…”

Mitochondrial and Cytosolic Isoforms of Yeast Fumarase Are Derivatives of a Single Translation Product and Have Identical Amino Termini

“…They suggested that these transcripts encode two fumarase products, one harboring a mitochondrial signal peptide and the other lacking this sequence, thereby encoding a cytosolic protein. In rat liver Tuboi and colleagues (15) proposed that the transcript of the single fumarase gene can be translated from two in-frame AUGs thereby giving rise to two products, one harboring and one lacking a mitochondrial signal peptide. Importantly both of the above alternative models of distribution predict the existence of two fumarase primary translation products.…”