Podcasts about apocytochrome

The question of whether cytochrome c could be functionally sorted to the mitochondrial intermembrane space along a "conservative sorting" pathway was investigated using a fusion protein termed pLc1-c. pLc1-c contains 3-fold targeting information, namely, the complete bipartite presequence of the cytochrome c1 precursor joined to the amino terminus of apocytochrome c. pLc1-c could be selectively imported into the intermembrane space either directly across the outer membrane along a cytochrome c import route or along a cytochrome c1 route via the matrix. Thus, apocytochrome c could be sorted along a conservative sorting pathway; however, following reexport from the matrix, apo-Lc1-c could not be converted to its holo counterpart. Despite the apparent similarity of structure and functional location of the heme lyases and similarity of the heme binding regions in their respective apoproteins, cytochrome c heme lyase and cytochrome c1 heme lyase apparently have different and nonoverlapping substrate specificities.

The cytochrome c import pathway differs markedly from the general route taken by the majority of other imported proteins, which is characterized by the import involvement of namely, surface receptors, the general insertion protein (GIP), contact sites and by the requirement of a membrane potential (Δψ). Unique features of both the cytochrome c precursor (apocytochrome c) and of the mechanism that transports it into mitochondria, have contributed to the evolution of a distinct import pathway that is not shared by any other mitochondrial protein analysed thus far. The cytochrome c pathway is particularly unique because i) apocytochrome c appears to have spontaneous membrane insertion-activity; ii) cytochrome c heme lyase seems to act as a specific binding site in lieu of a surface receptor and; iii) covalent heme addition and the associated refolding of the polypeptide appears to provide the free energy for the translocation of the cytochrome c polypeptide across the outer mitochondrial membrane.

The nuclear cyt-2-1 mutant of Neurospora crassa is characterized by a gross deficiency of cytochrome c (Bertrand, H., and Collins, R. A. (1978) Mol. Gen. Genet. 166, 1-13). The mutant produces mRNA that can be translated into apocytochrome c in vitro. Apocytochrome c is also synthesized in vivo in cyt-2-1, but it is rapidly degraded and thus does not accumulate in the cytosol. Mitochondria from wild-type cells bind apocytochrome c made in vitro from either wild-type or cyt-2-1 mRNA and convert it to holocytochrome c. This conversion depends on the addition of heme by cytochrome c heme lyase and is coupled to translocation of cytochrome c into the intermembrane space. Mitochondria from the cyt-2-1 strain are deficient in the ability to bind apocytochrome c. They are also completely devoid of cytochrome c heme lyase activity. These defects explain the inability of the cyt-2-1 mutant to convert apocytochrome c to the holo form and to import it into mitochondria.

Assembly of cytochrome c involves a series of steps: synthesis of apocytochrome c on free ribosomes, specific binding of apocytochrome c to the mitochondrial surface, transfer across the outer membrane, covalent addition of protoheme, refolding of the polypeptide chain, and association of holocytochrome c with its functional sites at the inner membrane. The binding step of apocytochrome c to Neurospora crassa mitochondria was studied by inhibiting the subsequent transfer steps with the heme analogue deuterohemin. The binding sites are highly specific for mitochondrial apocytochromes c. Bound labeled Neurospora apocytochrome c was competitively displaced by unlabeled apocytochrome c from various species. These exhibited different abilities for displacement. Apocytochrome c from Paracoccus denitrificans, the amino-terminal (heme-binding) fragment of Neurospora apocytochrome c, and Neurospora holocytochrome c did not recognize the binding sites. Polylysine did not interfere with apocytochrome c binding. Apocytochrome c is reversibly bound. The binding sites are present in limited number. High-affinity binding sites were present at about 90 pmol/mg of mitochondrial protein. They displayed an association constant of 2.2 X 10(7) M-1. Apocytochrome c was imported into mitochondria and converted to holocytochrome c directly from the binding sites when inhibition by deuterohemin was relieved. We conclude that the apocytochrome c binding sites on mitochondria represent receptors that function in the recognition and import of this precursor by mitochondria.

Transport of apocytochrome c across the outer mitochondrial membrane and conversion to holocytochrome c were studied in vitro. Apocytochrome c was synthesized in a cell-free homogenate from Neurospora crassa. Transfer in vitro was accomplished in a reconstituted system consisting of the postribosomal supernatant of the cell-free homogenate and of isolated and purified mitochondria from Neurospora. The reconstituted system has the following characteristics: * 1. Apocytochrome c is rapidly cleared from the supernatant and holocytochrome c appears in the mitochondria with the same kinetics. More than 80% of the apocytochrome c employed is converted to holocytochrome c. No transient accumulation of apocytochrome c is found in mitochondria. * 2. The heme group becomes covalently linked to apocytochrome c in the reconstituted system as demonstrated by analysis of tryptic peptide maps of the apoprotein and holoprotein. * 3. Deuterohemin added to the reconstituted system but not deuteroporphyrin inhibits the formation of holocytochrome c. This inhibition is reversed by protohemin. * 4. In the presence of deuterohemin about half of the apocytochrome c remains in the supernatant; the other half becomes associated with the mitochondria. The latter portion is tightly bound and is specifically released upon incubation of the mitochondria with excess apocytochrome c. It is converted to holocytochrome c after addition of protohemin. We conclude from these observations that apocytochrome c is transported across the outer mitochondrial membrane via receptor sites. In the presence of the heme analogue deuterohemin, binding to the receptor sites on the cytoplasmic surface of the outer mitochondrial membrane still takes place but translocation does not. The latter step is apparently coupled to the covalent linkage of the heme group. We suggest that the formation of the thioether bonds between apoprotein and heme is catalysed by an enzyme in the intermembrane space and that deuterohemin can compete with protohemin for binding to the enzyme. Finally, the data indicate that it is the heme group and not the porphyrin group which is coupled to the apoprotein.

# 1. Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. # 2. Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell-free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. # 3. Apocytochrome c synthesized in vitro was found in the post-ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labelling kinetics do not suggest a precursor role of microsomal apocytochrome c or holocytochrome c. # 4. Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. # 5. Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. # 6. The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The probles of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.