This study concerns the assembly into a multisubunit enzyme complex of a small hydrophobic protein imported into isolated mitochondria. Subunit 8 of yeast mitochondrial ATPase (normally a mitochondrial gene product) was expressed in vitro as a chimaeric precursor N9L/Y8‐1, which includes an N‐terminal‐cleavable transit peptide to direct its import into mitochondria. Assembly into the enzyme complex of the imported subunit 8 was monitored by immunoadsorption using an immobilized anti‐F1‐β monoclonal antibody. Preliminary experiments showed that N9L/Y8‐1 imported into normal rho+ mitochondria, with its complement of fully assembled ATPase, did not lead to an appreciable assembly of the exogenous subunit 8. With the expectation that mitochondria previously depleted of subunit 8 could allow such assembly in vitro, target mitochondria were prepared from genetically modified yeast cells in which synthesis of subunit 8 was specifically blocked. Initially, mitochondria were prepared from strain M31, a mit− mutant completely incapable of intramitochondrial biosynthesis of subunit 8. These mit− mitochondria however were unsuitable for assembly studies because they could not import protein in vitro. A controlled depletion strategy was then evolved. An artificial nuclear gene encoding N9L/Y8‐1 was brought under the control of a inducible promoter GALI. This regulated gene construct, in a low copy number yeast expression vector, was introduced into strain M31 to generate strain YGL‐1. Galactose control of the expression of N9L/Y8‐1 was demonstrated by the ability of strain YGL‐1 to grow vigorously on galactose as a carbon source, and by the inability to utilize ethanol alone for prolonged periods of growth. The measurement of bioenergetic parameters in mitochondria from YGL‐1 cells experimentally depleted of subunit 8, by transferring growing cells from galactose to ethanol, was consistent with the presence in mitochondria of a mosaic of ATPase, namely fully assembled functional ATPase complexes and partially assembled complexes with defective Fo sectors. These mitochondria demonstrated very efficient import of N9L/Y8‐1 and readily incorporated the imported processed subunit 8 protein into ATPase. Comparison of the kinetics of import and assembly of subunit 8 showed that assembly was noticeably delayed with respect to import. These findings open the way to a new systematic analysis of the assembly of imported proteins into multisubunit mitochondrial enzyme complexes.
|Number of pages||9|
|Journal||European Journal of Biochemistry|
|Publication status||Published - 1 Jan 1990|