Assembly of imported subunit 8 into the ATP synthase complex of isolated yeast mitochondria.
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-beta 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 GAL1. 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 F0 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.
Law
R H
RH
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Devenish
R J
RJ
Nagley
P
P
D000251:Adenosine Triphosphatases / Q000276:immunology; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005822:Genetic Vectors; D007163:Immunosorbent Techniques; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D009154:Mutation; D011498:Protein Precursors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000276:immunology / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D000911:Antibodies, Monoclonal; D011498:Protein Precursors; D011506:Proteins; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases