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The subcellular distribution of fumarase was investigated in the liver of various animals and in several tissues of the rat. In the rat liver, fumarase was predominantly located in the cytosolic and mitochondrial fractions, but not in the peroxisomal fraction. The amount of fumarase associated with the microsomes was less than 5% of the total enzyme activity. The investigation of the intracellular distribution of hepatic fumarase of the rat, mouse, rabbit, dog, chicken, snake, frog, and carp revealed that the amount of the enzyme located in the cytosol was comparable to that in the mitochondria of all these animals. The subcellular distribution of the enzyme in the kidney, brain, heart, and skeletal muscle of rat, and in hepatoma cells (AH-109A) was also investigated. Among these tissues, the brain was the only exception, having no fumarase activity in the cytosolic fraction, and the other tissues showed a bimodal distribution of fumarase in the cytosol and the mitochondria. The mitochondrial fumarase was predominantly located in the matrix. About 10% of the total fumarase was found in the outer and inner membrane, although it was unclear whether this fumarase was originally located in these fractions. No fumarase activity was detected in the intermembranous space.

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Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cellfree translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochonadria at 30 "C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Eur. J . Biochern. 126, 197-2021. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0°C for 5 min. When the incubation period at 0°C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30°C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).Porin is a major integral protein of the outer mitochondrial membrane, where it forms a channel for the non-specific passage of low-molecular-mass hydrophilic solutes [l]. Recently, this protein was purified from rat liver [2] and Neurospora crassa [3, 41 and its in vitro synthesis by this organism was reported [5 -71. Since porin can easily be synthesized in vitro in a cell-free translation system with RNA prepared from eukaryotic cells, it seems to be the most useful protein for use in studies on post-translational integration of proteins synthesized in cytoplasmic ribosomes into the outer mitochondrial membrane.In this paper, we report studies on the integration of porin, synthesized in a cell-free translation system, into the outer membrane of mitochondria from rat liver. The sequence of events involved in the integration of porin into the outer mitochondrial membrane is also discussed. MATERIALS AND METHODS AnimalsMale Wistar rats weighing about 200 g were used. Preparation of H-labeled porin and antibody against porinPorin was purified by the method of Freitag et al. 2 mg/ml) at 30°C. The mixture was treated with trypsin (final concentration, 250 Fg/ml) for 20 min at O"C, and the mitochondria were reisolated by centrifugation at 10000 x g for 5 min. The 35S-labeled porin was isolated by immunoprecipitation from the mitochondrial and post-mitochondria1 fraction and analyzed as described previously [ ...

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