A water-soluble form of porin from the mitochondrial outer membrane of Neurospora crassa

Mitochondrial porin, the outer membrane pore-forming protein, was isolated in the presence of detergents and converted into a water- soluble form. This water-soluble porin existed under nondenaturing conditions as a mixture of dimers and oligomers. The proportion of dimers increased with decreasing porin concentration during conversion. Water-soluble porin inserted spontaneously into artificial bilayers as did detergent-solubilized porin. Whereas the latter form had no specific requirements for the lipid composition of the bilayer, water- soluble porin inserted only into membranes containing a sterol, and only in the presence of very low concentrations of Triton X-100 (0.001% w/v) in the solution bathing the bilayer. The channels formed by water- soluble porin were indistinguishable from those formed by detergent- purified porin with respect to specific conductance and voltage dependence of conductance. Water-soluble porin bound tightly in a saturable fashion to isolated mitochondria. The bound form was readily accessible to added protease, indicating its presence on the mitochondrial surface. The number of binding sites was in the range of 5-10 pmol/mg of mitochondrial protein. Water-soluble porin apparently binds to a site on the assembly pathway of the porin precursor, since mitochondria whose binding sites were saturated with the water-soluble form did not import porin precursor synthesized in a cell-free system

Mitochondrial protein import

The precursor of F0-ATPase subunit 9 was bound to mitochondria in the absence of a mitochondrial membrane potential (delta psi). Binding was mediated by a protease-sensitive component on the mitochondrial surface. When delta psi was reestablished, bound precursor was directly imported without prior release from the mitochondrial membranes. A chimaeric protein consisting of the complete subunit 9 precursor fused to cytosolic dihydrofolate reductase (DHFR) was also specifically bound to mitochondria in the absence of delta psi. Two other fusion proteins, consisting either of the entire presequence of subunit 9 and DHFR or of part of the presequence and DHFR, were imported in the presence of delta psi. In the absence of delta psi, however, specific binding to mitochondria did not take place. We suggest that the hydrophobic mature part of subunit 9 is involved in the delta psi-independent binding of the subunit 9 precursor to receptor sites on the mitochondrial surface

Assembly of mitochondria

The majority of mitochondrial proteins are synthesized on cytoplasmic ribosomes and transferred to the mitochondria where they are assembled to supramolecular structures. The intracellular transfer of these proteins appears to occur by a post-translational mechanism, i.e., it involves extramitochondrial precursor forms which are translocated in a step independent from translation. The synthesis and transfer of individual proteins was investigated in vivo, or in vitro employing homologous and heterologous cell free systems for protein synthesis. Cytochrome c was initially made as the apoprotein. This precursor protein was converted to the holoprotein on uptake by mitochondria in reconstituted systems. Integrity of mitochondria was essential for the apo to holo conversion. In the case of the ADP/ATP carrier protein, an integral transmembrane protein of the inner mitochondrial membrane, the initial translation product had the same apparent molecular weight as the mature protein. It was found in soluble form in the post-ribosomal supernatant. Citrate synthase, a matrix protein, was synthesized as a precursor with an apparent molecular weight of 47 000. Transfer to the mitochondria was accompanied by cleavage to yield a molecular weight of 45 000. The significance of these results in relation to the mechanisms of intracellular transfer and of assembly of the individual proteins is discussed