A water-soluble form of porin from the mitochondrial outer membrane of Neurospora crassa. Properties and relationship to the biosynthetic precursor form.

R Pfaller,H Freitag,M A Harmey,R Benz,W Neupert

doi:10.1016/s0021-9258(17)39580-7

Abstract

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.

Highlights

Mitochondrial porin, the outer membrane poreforming protein, was isolated in the presence of detergents and converted into a water-soluble form
Water-soluble porin inserted spontaneously into artificialbilayers as did detergent-solubilized porin. Whereas the latter form had no specific requirements for the lipid composition of the bilayer, water-soluble porin inserted onlyinto membranes containing a sterol, and only in thepresence of very lowconcentrations of Triton X-100 (0.001%wfv) in the solution bathing the bilayer
Water-solubleporin 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

Summary

PROPERTIES AND RELATIONSHIPTOTHEBIOSYNTHETIC PRECURSOR FORM*

Mitochondrial porin, the outer membrane poreforming 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 precursor form of porin has been shown to insert into the outer membrane of mitochondria in vitro [14,15,16]. The soluble form of porin bindsto isolated mitochondria in a saturablefashion and competes with in vitro synthesized precursor for binding to and uptake into mitochondria and provides a model system to study precursor recognition and import into theouter mitochondrial membrane.

Preparation and Properties of isolated from mitochondria with

Highest conductance

DISCUSSION

Amino acid analysis of mature porin showed the presence

These sites may function in the recognition of the precursor

(1979) Methods