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...

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Male development in higher plants is a complex process which requires the correct spatial and temporal expression of a large number of male fertility genes. They include the genes required for the structure of the male organs, as well as genes required for male gamete development. Male-sterile mutants, impaired in male fertility functions, have helped to identify a number of these genes in various plant species including Arabidopsis thaliana, the model crucifer. In A. thaliana, once these genes are mapped, they can be cloned by chromosome walking. Alternative strategies of cloning will be facilitated by the isolation of similar mutants by tagging with transposable elements, T-DNA, or by mutagen-induced deletion. Once the genes required for male fertility are cloned and their wild type function identified, an understanding of the molecular basis of male fertility is likely to result. The combination of genetic dissection and the modern techniques of genome manipulation have made such a goal feasible.

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It has been documented that when furnished with an endomembrane signal sequence for the endoplasmic reticulum, beta-glucuronidase (GUS) is N-glycosylated, resulting in the nearly complete loss of enzymatic activity. To enable use of beta-glucuronidase as a reporter protein in secretory and vacuolar targeting studies, one of the two putative N-linked glycosylation sites within the GUS gene was altered by site-directed mutagenesis. The second N-linked glycosylation site was not altered because sequence analysis of nucleotide sequences around the second putative glycosylation site revealed that the published sequence was incorrect, and that no such site existed.