Functional reconstitution in Escherichia coli of the yeast mitochondrial matrix peptidase from its two inactive subunits.

The matrix processing peptidase from yeast (Saccharomyces cerevisiae) mitochondria was expressed in Escherichia coli via a plasmid-borne operon encoding the mature forms of the alpha and beta subunits of the enzyme. The subunits assembled into a fully active, soluble enzyme. The mature subunits were also expressed individually. The alpha subunit accumulated in large amounts and was obtained at a purity of 80% after a single chromatographic step. The beta-subunit-producing strain expressed an intact and a degraded form of the beta subunit, both of them soluble in the cytoplasm. Extract from either the alpha- or the beta-subunit-producing strain (S-alpha or S-beta extract, respectively), as well as the purified alpha subunit, was enzymatically inactive. However, precursor cleavage activity was restored by mixing either the S-alpha extract or the purified alpha subunit with the S-beta extract. The reconstituted processing activity was indistinguishable from the authentic holopeptidase.