Abstract
By use of anti-cytosolic fumarase antibody, a cDNA clone was isolated from a rat liver cDNA library in the expression vector lambda gt11 and in the pBR 322 vector. A clone with an insert of about 1.7 kilobases was isolated. Nucleotide sequence analysis of the insert revealed that the cDNA contained a 5'-noncoding region of 25 nucleotides, the coding region of 1,521 nucleotides, and a 3'-nontranslated region of 43 nucleotides followed by a poly(A)+ tail. The open reading frame encoded a polypeptide of 507-amino acid residues (predicted Mr = 54,462), which contained an additional sequence of 41-amino acid residues on the NH2 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 produced 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 obtained 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 and Southern blot, and primer-extension analyses using appropriate cDNA segments prepared with fumarase cDNA (1.7 kilobases) as a probe or primer suggested that the mitochondrial and cytosolic fumarases with identical amino acid sequences are encoded from a single gene and a possibility that the precursors of both these fumarases were synthesized by one species of mRNA having a base sequence coding the presequence of the mitochondrial fumarase by some unknown post-transcriptional mechanism(s).
Highlights
By use of anti-cytosolic fumarase antibodya, cDNA sis [1,2,3,4]
The open reading frame encoded a polypeptide of 507-amino acid residues, which contained an additional sequence of 41-amino acid residues on theNH2 terminus of the mitochondrial mature fumarase
To obtain more definite information on the primary structures of the fumarases and to clarify the mechanisms of their syntheses, we carried out molecular cloning of the cDNAs for the fumaramino acid sequence predicted from tnhuecleotide se- ases to determine their total amino acid sequences including quence contained all the amino acid sequences of 12 their precursor polypeptides
Summary
Amino acid sequencesare encoded from a single gene and a possibility that the precursors of both these fumarases were synthesized byonespecies of mRNA having a base sequence coding the presequence of the mitochondrial fumarase by some unknown post-transcriptional mechanism(s). Analyses of the Amino Acid Sequenceofs the NH2-terminal Portions Recent reports from our laboratory showed that rat liver of the Mitochondrial Fumarase and Proteolytic Peptides-The mitocontains twoisozymes of fumarate hydratase (EC 4.2.1.2) chondrial andcytosolic fumarases were each purified and crystallized which are localized in different intracellular compartments, the mitochondria and cytosol, and are almost distributed between these two subcellular compartments, and that these two fumarases cannot be differentiated from each other by any conventional methods such as immunochemical analyfrom rat liver by the method described previously [1]. Develosil ODS-5 was from Nomura Chemical Co. (Seto, Japan)
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