Translocation of proteins into rat liver mitochondria

Abstract

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 + .

Biosynthesis and processing of the large and small subunits of succinate dehydrogenase in cultured mammalian cells

“…1000-2000 and 4000-5000 higher than those of the corresponding mature forms. The data for the precursor to the large subunit is in agreement with the estimate obtained by Ono & Tuboi (1986) either in vitro by using rat liver mRNA to direct a cell-free translation system, or in vivo in ascites hepatoma cells. Our data concerning the biosynthesis of the small subunit provide the first demonstration that the Mr 27000 polypeptide is also synthesized as a higher-Me precursor.…”

“…The biosynthesis of SDH is of special interest since generation of the holoenzyme involves covalent attachment of FAD to the large subunit and insertion of the three Fe-S clusters. However, to date only one study on the biosynthesis of mammalian SDH, concerning the Mr 70000 subunit, has been reported (Ono & Tuboi, 1986). This paper describes the production and characterization of antisera to the bovine heart holoenzyme and to the individual large and small subunits.…”

Ornithine Aminotransferase, an Important Glutamate-Metabolizing Enzyme at the Crossroads of Multiple Metabolic Pathways

“…This is done using a signal peptide that is removed after translocation. Orientation toward the mitochondrion first occurs by the binding of the precursor to a receptor on the mitochondrial outer membrane; the signal peptide is removed after crossing this membrane [84]. As dealt with in detail in 2.1, there is some controversy about the exact length of this signal peptide.…”

Reconstitution and Physiological Protein Translocation Processes

“…Both the in vivo pulse-chase experiments (Hallermeyer and Neupert, 1976;Harmey et al, 1977) and in vitro experiments of uptake of some migratory proteins (Hallermeyer et al, 1977;Harmey et al, 1976) indicated such a post-translational event. Since these reports, numerous observations on post-translational translocation of mitochondrial proteins were made (e.g., see Argan et al, 1982;Chien et al, 1984;Gasser et al, 1982b;Hernandez-Yago et al, 1983;Kolansky et al, 1982;Lewin and Norman, 1983;Miralles et at., 1983;Mori et al, , 1985Nelson et at., 1979;Niranjan et at., 1988;Ono and Tuboi, 1986;Sakakibara et at., 1983Sakakibara et at., , 1987Suissa and Schatz, 1982;Yamamoto et at., 1981;Yoshida et at., 1985). During such studies it could generally be ascertained that post-translationally imported proteins reach the compartment where they are normally located (Campbell et at., 1982;Gasser et at., 1982a;Oda et at., 1984;Ono and Ito, 1984a,b;Ono and Tuboi, 1986).…”

“…Since these reports, numerous observations on post-translational translocation of mitochondrial proteins were made (e.g., see Argan et al, 1982;Chien et al, 1984;Gasser et al, 1982b;Hernandez-Yago et al, 1983;Kolansky et al, 1982;Lewin and Norman, 1983;Miralles et at., 1983;Mori et al, , 1985Nelson et at., 1979;Niranjan et at., 1988;Ono and Tuboi, 1986;Sakakibara et at., 1983Sakakibara et at., , 1987Suissa and Schatz, 1982;Yamamoto et at., 1981;Yoshida et at., 1985). During such studies it could generally be ascertained that post-translationally imported proteins reach the compartment where they are normally located (Campbell et at., 1982;Gasser et at., 1982a;Oda et at., 1984;Ono and Ito, 1984a,b;Ono and Tuboi, 1986). Post-translational translocation of proteins to chloroplasts was also reported in numerous cases (Chua and Schmidt, 1979;Cline et aI., 1985;Della-Cioppa et ai., 1986;Grossmann et ai., 1980Grossmann et ai., , 1982Highfield and Ellis, 1978;Minami et al, 1986;Pfisterer et al, 1982).…”