Alexander Tzagoloff

Assembly of the mitochondrial membrane system

SummaryNineteen mutants of S. cerevisiae exhibiting a double deficiency in cytochrome oxidase and coenzyme QH2-cytochrome c reductase (also cytochrome b deficient) have been studied. The mutants have been crossed to a set of ρ− tester strains with different segments of mitochondrial DNA. The mutants have also been crossed to mit− testers with defined genetic lesions. In addition, crosses were performed with a respiratory competent strain to ascertain whether mitotic and meiotic segregants could be isolated with only one of the two enzymatic deficiencies.The ρ− testers allowed the doubly deficient mutants to be separated into two classes. Mutants in class 1 were not restored by any of the ρ− testers and appeared to have separate mutations, one in cytochrome oxidase and the other in cytochrome b. Mutants in class 2 were restored by a set of ρ− clones whose retained segments of mitochondrial DNA contained the cytochrome b but not the cytochrome oxidase loci. These appeared to behave as single hit mutations. Further studies, however, indicated that both class 1 and class 2 mutants carried separate mutations in two different loci. Mitotic and meiotic segregants with a single enzymatic deficiency could be isolated. In a number of strains, the mutations were mapped in known cytochrome oxidase and cytochrome b loci. The apparent discrepancy of the ρ− tests for the class 2 mutants was shown to be probably due to a high unstability in one of the mutations.It has been concluded that all the doubly deficient strains carry two mutations in previously described cytochrome oxidase and cytochrome b loci. This conclusion argues against the existence of a single gene on mitochondrial DNA that controls the biosynthesis of the two respiratory enzymes.

High-expression vectors with multiple cloning sites for construction of trpE fusion genes : pATH vectors

Publisher Summary This chapter describes the structure and applications of a series of Escheriehia coli plasmids, the pATH plasmids, designed for the production of proteins from any cloned DNA sequence that contains an open reading frame (ORF). The cloned DNA sequences are fused in-frame to the trpE gene of E. coli, which codes for anthranilate synthase. Thus, the hybrid protein produced contains the amino-terminal 323 residues ofanthranilate synthase followed by the translation product specified by the cloned DNA. The advantages of pATH plasmids for production of hybrid proteins are as follows: (1) pATH plasmids are maintained in E. coli at high copy number. (2) The plasmids are relatively small, and their complete sequence is known. (3) Multiple cloning sites (MCS) are present following codon 323 of the trpE gene, for easy construction of in-frame gcne fusions. Different pATH plasmids carry MCS in each of the three registers of the translational reading frame.

Assembly of the mitochondrial membrane system XVI. Modified form of the ATPase proteolipid in oligomycin-resistant mutants of Saccharomyces cerevisiae.

Cytoplasmic mutations in Saccharomyces cerevisiae causing a resistance to 61igomycin have been shown to be associated with two genetically separate and unlinked loci on the mitochondrial DNA OLI 1 and OLI 2 [1,2]. It has been postulated that the mutations occur in gene products of the oligomycinsensitive adenosine triphosphatase [3]. This notion is substantiated by the finding that the ATPase of resistant strains is less sensitive to the inhibitor. To date, however, the lesion at the level of the protein components of the ATPase has not been identified. In this brief communication we report evidence showing a modification in OLI 1 resistant mutants of one of the mitochondrially synthesized subunits of the ATPase.

F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F1 ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F1, or the Atp11p and Atp12p chaperones that promote F1 assembly, have normal levels of the bicistronic ATP8/ATP6 mRNAs but fail to synthesize Atp6p and Atp8p. F1 mutants are also unable to express ARG8m when this normally nuclear gene is substituted for ATP6 or ATP8 in mitochondrial DNA. Translational activation by F1 is also supported by the ability of ATP22, an Atp6p-specific translation factor, to restore Atp6p and to a lesser degree Atp8p synthesis in the absence of F1. These results establish a mechanism by which expression of ATP6 and ATP8 is translationally regulated by F1 to achieve a balanced output of two compartmentally separated sets of ATP synthase genes.

Breakage of yeast: A method for processing multiple samples☆

Abstract A procedure is described for the rapid preparation of mitochondria and the soluble cell fraction of yeast. The method makes use of an adaptor for the Braun homogenizer which allows 16 samples to be processed at once.

The mapping of mutations in tRNA and cytochrome oxidase genes located in the cap-par segment of the mitochondrial genome of S. cerevisiae

Summary1.Two mutants have been isolated which carry mutations in mitochondrial tRNA genes; one in the aspartyl-tRNA gene and the other in one of the threonyl-tRNA genes. The mutant tRNAs are unable to be charged with their respective amino acids.2.These two mutations are located on the mitochondrial genome in the cap-par segment. Analyses of genetic recombination frequencies and co-retention and co-deletion frequencies of markers in petite strains yield an unambiguous gene order cap-asp-oxi 1-thr 1-oxi 2-par.3.Two loci involved in the specification of cytochrome oxidase (oxi1 and oxi2) show an average recombination frequency of 14% in pairwise crosses involving mutations of both loci. Although the two loci have a related function and are genetically linked they are shown to be separated by at least one tRNA gene.4.Pairwise intralocus crosses involving mit- mutations within either the oxi1 or oxi2 locus yield recombination frequencies <0.02–3.2%. However, no unique order could be derived for 19 oxi 1 and 6 oxi 2 mutations based on these data. In addition, the separation of mutant alleles within a single locus as the result of petite mutation was very rare. Consequently, attempts to order mutations within the locus from an analysis of the flanking markers in petites where separation occurred, provided only limited resolution.5.A discussion is presented of the limitations of the current mitochondrial genetic mapping techniques when applied to the fine resolution of glycerol negative mit- and syn- mutations.

SLC25A32 Mutations and Riboflavin-Responsive Exercise Intolerance

A patient with late-onset exercise intolerance had haploinsufficiency of SLC25A32, which encodes the human mitochondrial flavin adenine dinucleotide transporter. The patients symptoms were highly responsive to oral supplementation with riboflavin.

The Leader Peptide of Yeast Atp6p Is Required for Efficient Interaction with the Atp9p Ring of the Mitochondrial ATPase

Atp6p (subunit 6) of the Saccharomyces cerevisiae mitochondrial ATPase is synthesized with an N-terminal 10-amino acid presequence that is cleaved during assembly of the complex. This study has examined the role of the Atp6p presequence in the function and assembly of the ATPase complex. Two mutants were constructed in which the codons for amino acids 2–9 or 2–10 of the Atp6p precursor were deleted from the mitochondrial ATP6 gene. The concentration of Atp6p and ATPase complex was approximately 2 times less in the mutants. The lower concentration of ATPase complex in the leaderless mutants correlated with less Atp6p complexed with the Atp9p ring of the F0 sector and with accumulation of an Atp6p-Atp8p complex that aggregated into polymers destined for eventual proteolytic elimination. We propose that the presequence either targets Atp6p to the Atp9p or signals insertion of the Atp6p precursor into a microcompartment of the membrane for more efficient interaction with the Atp9p ring. Despite the ATPase deficiency, growth of the leaderless atp6 mutants on respiratory substrates and the efficiency of oxidative phosphorylation were similar to that of wild type, indicating that the mutations did not affect the proton permeability of mitochondria.

[42] Oligomycin-sensitive ATPase of Saccharomyces cerevisiae

Publisher Summary The oligomycin-sensitive ATPase complex of yeast mitochondria is analogous to the ATPase complex of mammalian mitochondria and consists of three functionally distinct components: (1) a water-soluble and oligomycin-insensitive ATPase called “F 1 ,” (2) a membrane factor consisting of four subunit proteins that confer oligomycin sensitivity on F 1 , and (3) oligomycin-sensitivity-conferring protein (OSCP), which functions in the attachment of F 1 to the membrane factor. Both F 1 and the oligomycin-sensitive ATPase complex have been purified from mitochondria of Saccharomyces cerevisiae . Methods have been devised for the purification of the subunit proteins of F 1 . This chapter provides an overview on the purification of F 1 subunits. The two major and one of the minor subunits of yeast F 1 are purified by a modification of procedures previously used to purify the subunits of F 1 from bovine heart and rat liver mitochondria. The entire procedure is carried out at room temperature.

[4] Ubiquinol-cytochrome-c oxidoreductase from Saccharomyces cerevisiae

Publisher Summary This chapter focuses on the ubiquinol-cytochrome-c oxidoreductase from saccharomyces cerevisiae . As a consequence, such strains have to be identified among the broader class of respiratory-deficient nuclear petite (pet) mutants. The chapter discusses the screening for cytochrome-deficient mutants. Mutations are transferred to the strains of opposite mating type to that of the original pet isolates by crosses to a respiratory competent haploid with complementing auxotrophic markers. Following sporulation, the meiotic progeny are checked for the desired mating type, auxotrophy, and the presence of the pet mutation either by tetrad or random spore analysis. The chapter also describes the status of cytochrome b. All the nuclear genes for the constituent polypeptides of the yeast complex have been cloned and natural mutants or construct with null mutations in each gene are available.