Rat liver mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single mRNA with two alternative in-phase aug initiation sites

Abstract

By use of anticytosolic fumarase antibody, a cDNA clone was isolated from a rat liver cDNA library in the expression vector λgt11 and in the pBR 322 vector. A clone with an insert of about 1.7 kbp was isolated. Nucleotide sequence analysis of the insert revealed that the cDNA contained a noncoding region composed of 25 nucleotides in the 5′ terminus, the coding region composed of 1,521 nucleotides, and the 3′ nontranslated region composed of 43 nucleotides followed by a poly(A) + tail. The open reading frame encoded a polypeptide of 507 amino acid residues (predicted M r = 54,462), which contained an additional sequence composed of 41 amino acid residues on the N-terminus of the mitochondrial mature fumarase (the presequence). Thus, this reading frame was concluded to encode the precursor of mitochondrial fumarase. The amino acid sequence predicted from the nucleotide sequence contained all the amino acid sequences of 12 proteolytic polypeptides obtained by digestion of purified mitochondrial fumarase with V8 protease. The total amino acid sequence of the mitochondrial fumarase also contained all the sequences of 14 proteolytic peptides prepared from the cytosolic fumarase, indicating that the amino acid sequences of these two isozymes are identical. Furthermore, the results obtained by hybrid-selected translation, Northern blot and primer-extension analyses using appropriate cDNA segments prepared from fumarase cDNA (1.7 kbp) as the probe or primer suggested a possibility that both precursors of the mitochondrial and cytosolic fumarases were synthesized with one species of mRNA having base sequence coding presequence of the mitochondrial fumarase by unknown post-transcriptional mechanism(s). Rat liver cells may contain a specific RNA(18S) modulating the translational activity of mRNA for fumarase. This RNA(s), which was contained in poly(A) − fraction, was partially purified by high-performance gel filtration. The partially purified RNA(s) suppressed the translational activity of the cytosolic fumarase, whereas the translational activity of the mitochondrial one was accelerated by this RNA(s).