title: Functional reconstitution in Escherichia coli of the yeast mitochondrial matrix peptidase from its two inactive subunits.
abstract: 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.
authors.affiliation: Laboratoire d'Ingénierie et de Dynamique des Systèmes Membranaires, GDR1000, Centre National de la Recherche Scientifique, Marseille, France.
authors.forename: V
authors.initials: V
authors.lastname: Géli
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D000096122:Mitochondrial Processing Peptidase
chemical_list: D008666:Metalloendopeptidases; D009838:Oligodeoxyribonucleotides; D000096122:Mitochondrial Processing Peptidase