Mechanism of synthesis and localization of mitochondrial and cytosolic fumarases in rat liver

Abstract

Fumarases in the mitochondrial and cytosolic fractions of rat liver were separately purified and crystallized. These two fumarases were not distinguishable in physicochemical, catalytic, or immunochemical properties. The sequences of seven amino acids in the C-terminal portions of the two fumarases were shown using carboxypeptidase P to be identical, i.e.-Val-Asp-Glu-Thr-Ala-Leu-Lys-. The amino acid sequence of the N-terminal portion of the mitochondrial fumarase was determined by the Edman method as Ala-Gln-Gln-Asn-Phe-Glu-Ile-Pro-Asp-, but that of the cytosolic fumarase could not be determined by the Edman method, since the N-terminal amino acid was blocked. The N-terminal amino acid of the cytosolic fumarase was identified as N-acetyl-alanine by analysis of the acidic amino acid produced by digestion of the enzyme protein with pronase E, carboxypeptidase A and B. Then the sequence of five amino acids in the N-terminal portion was determined by analyzing the acidic peptide obtained by limited proteolysis of the enzyme protein with carboxypeptidase A as Ac-Ala-Ser-Gln-Asn-Ser-. Peptide mapping of the tryptic peptides obtained from the mitochondrial and cytosolic fumarases showed no difference in the amino acid sequences of the two except in their N-terminal portions. The turnover rates of the mitochondrial and cytosolic fumarases were determined by injecting l-[U- 14C]leucine into rat and following the decay of specific radioactivity incorporated into immunoprecipitates from the partially purified enzyme. The half-life of the cytosolic fumarase was estimated as 4.8 days from the decay curve of its specific radioactivity. The decay curve of the specific radioactivity of the mitochondrial fumarase, obtained after a single injection of l-[U- 14]leucine, was quite unusual: its specific radioactivity remained constant for about 7 days after pulse labeling, and then decreased exponentially with a half-life of 9.7 days. Similar amounts of cytosolic and mitochondrial fumarase were found in the livers of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp, respectively. Similar subcellular distributions of the enzyme were also found in the kidney, heart, and skeletal muscle of rats, and in hepatoma cells (AH-109A). However, in rat brain no fumarase activity was detected in the cytosolic fraction. Two putative precursor polypeptides of rat liver fumarase were synthesized when rat liver RNA was translated in vitro in a rabbit reticulocyte lysate system. One of these putative precursor polypeptides (P 1) had a molecular mass of about 5,000 daltons more than the mature subunit of fumarase (45,000 daltons), while the other (P 2) had the same molecular mass as the mature enzyme. When the 35S-labeled cell-free translation products were incubated with rat liver mitochondria at 30°C, P 1 and the 35S-labeled mature size fumarase became associated with mitochondria. The 35S-labeled mature size fumarase was resistant to externally added protease, but P 1 was not, indicating that the 35S-labeled mature size fumarase was located in the mitochondrial matrix. The following observations strongly suggested that the 35S-labeled mature size fumarase in mitochondria was derived from P 1, which was imported energy-dependently and concomitantly processed to the mature size. 1) The amount of the 35S-labeled mature size fumarase recovered from the mitochondria increased with the duration of incubation, while the amount of P 1 recovered from the post-mitochondrial and mitochondrial fractions decreased with the duration of incubation. 2) Only P 1 could bind with the mitochondrial outer membrane at 0°C even in the presence of an uncoupler of oxidative phosphorylation. 3) P 1 bound to the mitochondrial outer membrane at 0°C was imported into the matrix, when the mitochondria were reisolated and incubated at 30°C in the presence of an energy-generating system. These results suggest that the precursor of the mitochondrial enzyme has a larger molecular weight than the mature enzyme, whereas the precursor for the cytosolic enzyme has the same molecular weight as the mature enzyme and that these two precursors of fumarase are coded by two different mRNAs.