Reconstitution of Import-Competent Outer Membrane Vesicles from Mammalian Mitochondria1

Abstract

Protein insertion into mitochondrial outer membrane (OM) vesicles isolated from Neurospora crassa has recently been reported. The N. crassa OM vesicles retained the features of the intact mitochondria concerning 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 proteoliposomes were reconstituted from OM vesicles using heptyl beta-thioglucoside. Both OM vesicles and the reconstituted outer membrane vesicles (ROM) were able to import porin, but unable to import the precursor of adrenodoxin, which translocates across both the outer and inner membranes of intact mitochondria. Porin insertion into both OM vesicles and ROM was inhibited in the presence of purified recombinant adrenodoxin precursor and also by ATP depletion, and was dependent on the trypsin-sensitive membrane surface factor, suggesting that the purified OM vesicles as well as ROM retained the properties of the intact OM concerning porin insertion. The protein import machinery of OM seems to be functional for the outer membrane protein without the participation of the inner membrane. The successful reconstitution of the protein import activity from solubilized OM will pave the way for further biochemical characterization of the protein import machinery of OM.