RECONSTITUTION OF IMPORT-COMPETENT OUTER-MEMBRANE VESICLES FROM MAMMALIAN MITOCHONDRIA

Protein insertion into mitochondrial outer membrane (OM) vesicles isol ated from Neurospora crassa has recently been reported. The N. crassa OM vesicles retained the features of the intact mitochondria concernin g the dependency of insertion on the receptor protein [A. Mayer et al. (1993) J. Cell Biol. 121, 1233-1243]. In this study, OM vesicles were purified from bovine adrenal cortex mitochondria, and unilamellar pro teoliposomes were reconstituted from OM vesicles using heptyl beta-thi oglucoside. Both OM vesicles and the reconstituted outer membrane vesi cles (ROM) were able to import porin, but unable to import the precurs or of adrenodoxin, which translocates across both the outer and inner membranes of intact mitochondria. Porin insertion into both OM vesicle s and ROM was inhibited in the presence of purified recombinant adreno doxin precursor and also by ATP depletion, and was dependent on the tr ypsin-sensitive membrane surface factor, suggesting that the purified OM vesicles as well as ROM retained the properties of the intact OM co ncerning porin insertion. The protein import machinery of OM seems to be functional for the outer membrane proteins without the participatio n of the inner membrane. The successful reconstitution of the protein import activity from solubilized OM will pave the way for further bioc hemical characterization of the protein import machinery of OM.