Hideyu Ono

Presence of the cytosolic factor stimulating the import of precursor of mitochondrial proteins in rabbit reticulocytes and rat liver cells

Previously we purified a cytosolic factor that stimulates the import of the extrapeptide (the synthetic peptide of the presequence of ornithine aminotransferase) into the mitochondrial matrix (Ono, H., and Tuboi, S., 1988, J. Biol. Chem. 263, 3188-3193). In this work this cytosolic factor was shown also to stimulate the import of the precursors of ornithine aminotransferase, a large subunit of succinate dehydrogenase, and sulfite oxidase. The amounts of these precursors bound to the outer mitochondrial membrane were increased by this cytosolic factor, suggesting that the cytosolic factor participates in the recognition step in the import process of the precursor protein. When the cytosolic factor was applied to an ATP-agarose column, the import-stimulating activity was recovered entirely in the unadsorbed fraction. Immunochemical studies showed that in these conditions the 70-kDa heat shock-related protein (Hsp 70) was present exclusively in the fraction adsorbed to the ATP-agarose column. The cytosolic factor is thus different from the 70-kDa heat shock-related protein, which was identified as a factor required for the import of mitochondrial proteins in yeast. The cytosolic factor was also detected in the cytosol of rat liver cells, and a considerable amount of this factor was recovered from rat liver mitochondria by washing them with high salt buffer, suggesting that the cytosolic factor has affinity to the outer mitochondrial membrane and binds to its receptor on the membrane. From these results, we conclude that the cytosolic factor forms a complex with the precursor of mitochondrial protein and then this complex binds to the outer mitochondrial membrane, probably via the receptor of the cytosolic factor.

Impaired activation of caspase cascade during cell death induced by newly synthesized singlet oxygen generator, 1-buthylnaphthalene-4-propionate endoperoxide.

Endoperoxides of naphthalene derivatives generate singlet oxygen under physiological conditions. Here we have synthesized a new endoperoxide of a naphthalene derivative, 1‐buthylnaphthalene‐4‐propionate endoperoxide (BNPE), and studied its cytotoxic properties on HepG2 and HaCaT cells. BNPE induced cell death at much lower concentration than 1‐methylnaphthalene‐4‐propionate endoperoxide (MNPE) and naphthalene dipropionate endoperoxide (NDPE). A positive correlation exists between the amount of endoperoxide incorporated into cells and its cytotoxic ability. The cytotoxic effect of BNPE was attenuated by α‐tocopherol but not by sodium azide. In contrast, the effects of MNPE and NDPE were attenuated by both α‐tocopherol and sodium azide. The caspase cascade in cells treated with endoperoxide was impaired. Caspase activity in a soluble protein fraction were inhibited similarly by the above three endoperoxides. These results suggest an abortive apoptotic pathway due to the suppression of caspase activation is a general feature of cell death induced by singlet oxygen.

Purification of 52 kDa protein : a putative component of the import machinery for the mitochondrial protein-precursor in rat liver

A protein having a molecular mass of 52 kDa was purified to homogeneity from solubilized mitochondrial membrane proteins by affinity column chromatography using the synthetic presequence of ornithine aminotransferase (OAT) as the ligand. This 52 kDa protein was specifically bound to the affinity column and eluted with 1 mM OAT-presequence, indicating that it recognized the presequence and bound to it specifically. Anti-52 kDa protein Fab fragments specifically inhibited the import of OAT-precursor into mitochondria, showing that the 52 kDa protein plays an essential role in this process. These results suggest that 52 kDa protein is a component of the import machinery of the mitochondrial protein-precursor in the mitochondrial membrane.

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

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.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for intra-mitochondrial degradation of the extrapeptide of ornithine aminotransferase

When rat liver mitochondria that had imported a synthetic extrapeptide of ornithine aminotransferase (composed of 34 amino acids) were incubated at 25 degrees C, the extrapeptide in their matrix was degraded inside the mitochondria. The degradation of the extrapeptide did not depend on energy either inside or outside the mitochondria. The degrading activity was found exclusively in the mitochondrial soluble fraction and only inhibited by N-ethylmaleimide of eight protease-inhibitors tested. These observations show that the extrapeptide cleaved from the precursor of the mitochondrial protein in the mitochondria is degraded by some ATP-independent proteases inside the mitochondria.

Transport of prepro-albumin into inverted vesicles prepared from the inner membrane of rat liver mitochondria

When inverted vesicles prepared from the inner membrane of rat liver mitochondria were incubated with prepro‐rat serum albumin, considerable amounts of prepro‐albumin and pro‐albumin were recovered with the inverted vesicles re‐isolated by centrifugation. Pro‐albumin was resistant to trypsin, but prepro‐albumin was completely digested by trypsin, indicating that prepro‐albumin was transported into the vesicles and concomitantly converted to pro‐albumin. This transport process required ATP, but not a membrane potential. These results suggest that some export machinery for a protein having an amino acid sequence in its N‐terminal portion similar to the signal sequence of secretory protein exists in the inner mitochondrial membrane.