A mitochondrial gene from Saccharomyces cerevisiae encoding a hydrophobic membrane protein, subunit 8 of the FO/Fl-type mitochondrial ATPase complex, has been functionally replaced by an artificial nuclear gene specifying an imported version of this protein. The experiments reported here utilized a multicopy expression vector (pLF1) that replicates in the nucleus of yeast cells and that carries an inserted DNA segment, specifying a precursor protein (N9/Y8) consisting of subunit 8 fused to an N-terminal cleavable transit peptide (the leader sequence from Neurospora crassa ATPase subunit 9). The successful incorporation of the imported subunit 8 into functional ATPase complexes after transformation with pLF1 expressing N9/Y8 was indicated by the efficient genetic complementation of respiratory growth defects of aapl mit-mutants, which lack endogenous subunit 8. The reconstitution of ATPase function was confirmed by biochemical assays of ATPase performance in mitochondria and by immunochemical analyses that demonstrated the assembly of the cytoplasmically synthesized subunit 8 into the ATPase complex. Reconstitution of ATPase function required the cytoplasmically synthesized subunit to have a transit peptide. The strategy for importation and reconstitution developed for subunit 8 leads to a systematic approach to the directed manipulation of mitochondrially encoded membraneassociated proteins that has general implications for exploring membrane biogenesis mechanistically and evolutionarily.Considerable insight into the assembly and function of energy-transducing complexes of microorganisms has been derived from molecular genetic studies (1). The FO/F1-type mitochondrial ATPase (mtATPase) of Saccharomyces cerevisiae is such a complex, in which the three intensely hydrophobic proteins of the FO membrane sector have been subjected to extensive genetic and biochemical analyses (2, 3). The three proteins, subunits 6, 8, and 9, are encoded by yeast mtDNA. A multifaceted approach has been applied, including mutational analysis by gene sequencing and detailed investigation of the mutant phenotypes at physiological and molecular levels. The data obtained have begun to define the roles of individual amino acid residues and domains of these proteins in their interactive assembly into the membrane, in the function of the proton channel of FO, and in energy-coupling leading to ATP synthesis catalyzed by the soluble F1 sector of the complex. Thus it has been shown that both transmembrane stems (4) and a hydrophilic charged loop (5) of subunit 9 (76 amino acids), together with two of the five or more transmembrane stems (6) of subunit 6 (259 amino acids), participate in protonophoric functions and energy coupling (3). Studies on protein assembly (7) have shown that subunit 8, a 48-amino acid polypeptide (8) with a single transmembrane stem (9), is required for the assembly of subunit 6 into the mtATPase complex, while the assembly of subunit 8 is dependent upon the correct integration of subunit 9 into the membrane (2).A...

This paper deals with a truncated derivative of subunit 8 of yeast mitochondrial ATP synthase in which a conserved positively charged residue (Lys47) has been removed by site-directed mutagenesis together with the Cterminal residue (Leu48). This derivative has been expressed as a chimaeric precursor N9L/Y8-1 (K47-STP) carrying an N-terminal cleavable leader sequence (N9L), fused by a short bridging sequence to the truncated subunit-8 passenger protein. Allotopic expression of N9L/YS-l(K47-STP) in vivo in an aapl mit-host yeast strain lacking endogenous subunit 8 leads to partial restoration of bioenergetic function in the transformant strain denoted T475. Import and assembly studies were carried out in vitro using target mitochondria from strain YGL-1 partially depleted in subunit 8 ; such controlled depletion has been previously shown to be required for the efficient assembly (monitored immunochemically) of full-length subunit 8 imported in vitro as the precursor N9L/Y8-1. It was found that N9L/YS-l(K47-STP) synthesized in vitro was imported successfully into YGL-1 mitochondria, but no significant assembly of the truncated subunit 8 was observed in these or any other mitochondria tested. The bioenergetic defects in T475 mitochondria are ascribed to the impaired assembly of the subunit-8 variant in vivo, resulting from the truncation at Lys47. In consequence, T475 mitochondria behave as though partially depleted of subunit 8. This conclusion was supported by the ability of isolated T475 mitochondria to provide a vehicle for the efficient import and assembly of subunit 8 processed form full-length N9L/Y8-1. Two related aspects of import and assembly have been addressed as part of the analysis of truncated subunit 8. First, mitochondria from strain T2-1, an aapl mit-mutant genetically reconstituted by allotopic expression of N9L/ Y8-1, were also found to be effective in the in vitro assembly of subunit 8 derived from imported N9L/Y8-1. This suggests an intramitochondrial shortage of subunit 8 delivered by allotopic expression of N9L/Y8-1 in vivo, which may underlie the incomplete restoration of energy coupling in T2-1 mitochondria compared to those of wild-type yeast. Second, on allotopic expression of N9L/Y8-2 (containing subunit 8 directly fused to N9L) in the aapl mithost, a rescued transformant strain T10-1 was generated which displays bioenergetic defects superficially similar to those of T475. Processed subunit 8 clearly assembled into the ATP synthase of isolated YGL-1 mitochondria, in spite of the relatively weak import of N9L/Y8-2 in vitro. This demonstrates the ability of the in vitro assembly system to distinguish assembly properties of variants of subunit 8, such as N9L/YS-l(K47-STP) and N9LIY8-2.The Fa sector of the yeast mitochondrial ATP synthase (mtATPase) provides a versatile system for the study of the assembly of integral membrane proteins into energy-transducing enzyme complexes [l]. Previous studies of mutants of Saccharomyces cerevisiae, affected in the three mitochondrial genes encoding Fa s...

Direct fusions have been constructedbetween each of subunits 8 and 9 from mitochondrial ATPase of Saccharomyces cerevisiae, proteins normally encoded inside mitochondria, and the cleavable N-terminal transit peptide from the nuclearly encoded precursor to subunit 9 of Neurospora crassa mitochondrial ATPase. The subunit 8 construct was imported efficiently into isolated yeast mitochondria and was processed at or very near the fusion point. When expressed in vivo from its artificial nuclear gene, this cytoplasmically synthesized form of subunit 8 restored the growth defects of aapl mutants unable to produce subunit 8 inside the mitochondria.The subunit 9 construct was, however, unable to be imported into isolated mitochondria and could not, following nuclear expression in vivo, complement growth defects in mitochondrial olil mutants. This behaviour is contrasted with the previously demonstrated import competence of another yeast subunit 9 fusion, bearing the first five residues of mature N. crassa subunit 9 interposed between its own transit peptide and the yeast subunit 9 moiety.

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The function of the positively charged C‐terminal region of mitochondrially encoded subunit 8 of yeast mitochondrial ATP synthase was investigated using derivatives truncated at each of the 3 positively charged residues (Arg37, Arg42 and Lys47). Each construct, allotopically expressed in the nucleus, was tested for its ability to import and assemble functionally into ATP synthase in yeast cells unable to synthesize mitochondrial subunit 8. The efficiency of import of each construct into isolated wild‐type yeast mitochondria was also determined. One construct truncated at the penultimate residue of subunit 8 (Lys47) functions in vivo and shows efficient import in vitro. Thus subunit 8 can function with only two positively charged residues. The remainder of the subunit 8 variants failed to rescue in vivo. Since they all show greatly reduced or undetectable import in vitro, presumably because of the increased hydrophobic character of the subunit 8 moiety in the chimaeric precursors, the status of these variants as regards assembly and function is not clear.