Cytochrome b2 is sorted into the intermembrane space of mitochondria by a bipartite N‐terminal targeting and sorting presequence. In an attempt to define the sorting pathway we have identified an as yet unknown import intermediate. Cytochrome b2‐dihydrofolate reductase (DHFR) fusion proteins were arrested in the presence of methotrexate (MTX) so that the DHFR domain was at the surface of the outer membrane while the N‐terminus reached into the intermembrane space where the sorting signal was removed. This membrane‐spanning, mature‐sized species was efficiently chased into the mitochondria upon removal of MTX. Thus, an intermediate was generated which was exposed to the intermembrane space but was still associated with the inner membrane. This intermediate was also found upon direct import of cytochrome b2 and derived fusion proteins. These membrane‐bound mature‐sized cytochrome b2 species loop through the matrix and could be recovered in a complex with mt‐Hsp70 and the inner membrane MIM44/ISP45, a component of the inner membrane import apparatus. This novel sorting intermediate can only be explained by a pathway in which cytochrome b2 passes through the matrix. The existence of such an intermediate is inconsistent with a pathway by which entrance of the mature part of cytochrome b2 into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with the conservative sorting pathway.

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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 [ ...

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic mature enzyme when rat liver total RNA was translated in a cell-free system. When the in vitro translation products were incubated with isolated rat liver mitochondria, pre-sulfite oxidase was recovered in mitochondria and converted to the size of the mature enzyme. The in vitro-processed mature enzyme was recovered in the intermembrane space of mitochondria (Ono, H. & Ito, A. (1981) Biochem. Biophys. Res. Commun. 107, 258-264). Mature sulfite oxidase was not imported into mitochondria, and did not affect the import of pre-sulfite oxidase. When mitochondria were incubated with gel-filtered translation products, the import was dependent on ATP, and the activity restored by the addition of ATP was blocked by valinomycin and K+ ion. These results suggest that the import of pre-sulfite oxidase into mitochondrial intermembrane space requires an electrochemical potential across the inner membrane. When mitochondria were fractionated, most of the processing activity was recovered in the mitoplast, whereas the inner membrane (after being mostly inverted by sonication) exhibited only slight activity. The processing activity was strongly inhibited by some metal chelators including EDTA, GTP, and Zincon. It was not inhibited by phenyl methyl sulfonyl fluoride, aprotinin, or various microbial protease inhibitors including pepstatin, antipain, leupeptin, and chymostatin. The processing enzyme seems to be a metal protease. The processing of pre-sulfite oxidase by mitoplasts was energy-dependent.

The precursor polypeptides of a large subunit of succinate dehydrogenase and ornithine aminotransferase (the enzymes which are located in the mitochondrial inner membrane and matrix respectively) were synthesized as a larger molecular mass than their mature subunits, when rat liver RNA was translated in vitro. These precursor polypeptides were also detected in vivo in ascites hepatoma cells (AH-1 30 cells). When the 35S-labeled precursor polypeptides were incubated with isolated rat liver mitochondria at 30 "C in the presence of an energy-generating system, these two precursors were converted to their mature size and the 35S-labeled mature-size polypeptides associated with mitochondria. Furthermore, these mature-size polypeptides were recovered from their own locations, the inner mitochondrial membrane and the matrix.The precursor of ornithine aminotransferase incubated with rat liver mitochondria at 0 "C was specifically and tightly bound to the surface of the mitochondria even in the presence of an uncoupler of oxidative phosphorylation. This precursor, bound to the mitochondria, was imported into the matrix when the mitochondria were reisolated and incubated at 30 "C in the presence of an energy-generating system, suggesting that a specific receptor may be involved in the binding of the precursor.The processing enzyme for both precursor polypeptides seemed to be located in the mitochondrial matrix and was partially purified from the mitochondria. A metal-chelating agent strongly inhibited the processing enzyme and the inhibition was recovered by the addition of MnZ+ or C o 2 + .