On the formation of rho- petites in yeast. II. Effects of mutation tsm-8 on mitochondrial functions and rho-factor stability in Saccharomyces cerevisiae.

1. In non-fermentable substrates growth of mutant tsm-8 cells of Saccharomyces cerevisiae is restricted to about one generation after shift from 23 to 35 degrees C. Non-permissive conditions (35 degrees C, glycerol) cause a gradual decrease in respiration to about 20% of the activity at permissive temperature 23 degrees C). 2. Anaerobically grown and glucose-repressed mutant cells exhibit a decreased adaptation rate of mitochondrial functions to aerobic growth and non-fermentative growth, even at 23 degrees C, as revealed by determination of respiratory rates and mitochondrial protein synthesis. 3. At 35 degrees C, rho+ cells of mutant tsm-8 are converted to p- cells within 6-8 generations of growth, in all fermentable substrates tested. Drugs or antibiotics as nalidixic acid, acriflavin, chloramphenicol and erythromycin, bongkrecic acid, antimycin and FCCP, as well as anaerobiosis, have little or no influence on this kinetics. A heat shock does not yield rho- petites to a significant extent. 4. Reversion of tsm-8 cells to wild type function, which occurs spontaneously with a frequency of 10(-8), is found to be due to a mitochondrial mutational event.     

Spontaneous and induced rho mutants of Saccharomyces cerevisiae: patterns of loss of mitochondrial genetic markers.

The deletion which leads to spontaneous rho mutants occurs preferentially at a unique region covering genes oxi3, pho1/OII, and mit175. The frequency of loss of genetic markers in this region was significantly higher than in other regions as determined with a 15- marker system. When various mutagenic treatments were applied, this specific pattern of deletion was also observed, but it was dramatically amplified. This suggests that the basic mechanism of rho production is the same in yeast mitochondrial genomes in both spontaneous and induced mutants.     

A temperature sensitive mitochondrial mutation of Saccharomyces cerevisiae.

A mitochondrially inherited temperature sensitive respiratory deficient mutant of yeast has been isolated. Detection of nuclear suppressor mutations indicates an interaction between the nuclear and mitochondrial genomes. A preliminary biochemical characterization is presented.     

Production of petites by cell cycle mutants of Saccharomyces cerevisiae defective in DNA synthesis.

Summary Mutations in two genes (cdc8 and cdc21) required for nuclear and mitochondrial DNA synthesis in Saccharomyces cerevisiae result in a 6- to 11-fold increase in the rate of mitotic segregation of petites at the permissive temperature. The defect in DNA replication and the increased rate of petite production result from the same mutation since the two phenotypes cosegregate and corevert. Most of the petites isolated from strains carrying mutations in cdc8 and cdc21 contain mtDNA. Therefore, the petites do not result simply from an underreplication of mitochondrial DNA. The mutation rates for nuclear and mitochondrial genes are the same in cdc8, cdc21 and their wild-type parent. Therefore the petites are unlikely to result from an increase in the rate of base pair substitution.     

The differential overamplification of short sequences in the mitochondrial DNA of rho- petites in Saccharomyces cerevisiae stimulates recombination

It is well known that the yeast Saccharomyces cerevisiae can be affected by mutations, called 'petite colonie', which correspond to the loss of the major part of the mitochondrial DNA and the concomitant amplification of the remaining sequence, the basic repeat unit (BRU). We describe here a new phenomenon, the internal overamplification (IOA), due to the differential amplification (up to 20-fold) of short sequences within the BRU. These IOAs are very stable and stimulate the recombination. We discuss here the possible mechanisms giving rise to the appearance and maintenance of the IOAs within the BRU and their effect on the recombination process.     

Ascospore formation in the yeast Saccharomyces cerevisiae.

Sporulation of the baker's yeast Saccharomyces cerevisiae is a response to nutrient depletion that allows a single diploid cell to give rise to four stress-resistant haploid spores. The formation of these spores requires a coordinated reorganization of cellular architecture. The construction of the spores can be broadly divided into two phases. The first is the generation of new membrane compartments within the cell cytoplasm that ultimately give rise to the spore plasma membranes. Proper assembly and growth of these membranes require modification of aspects of the constitutive secretory pathway and cytoskeleton by sporulation-specific functions. In the second phase, each immature spore becomes surrounded by a multilaminar spore wall that provides resistance to environmental stresses. This review focuses on our current understanding of the cellular rearrangements and the genes required in each of these phases to give rise to a wild-type spore.     

Effect on gluconeogenesis of mutants blocking two mitochondrial transport systems in the yeast Saccharomyces cerevisiae

Two mutants of Saccharomyces cerevisiae, ccr1 and tpy1, have been found to interfere with the transport of small molecules across the inner mitochondrial membrane. Both also have the effect of interfering with the synthesis of a number of cytoplasmically located enzymes involved in gluconeogenesis, even when the cells are released from glucose repression. The ccr1 mutant, defective in the transport of dicarboxylic acids across the inner membrane, represses the synthesis of gluconeogenic enzymes almost totally, but synthesis can be induced on complete medium without a carbon source. This mutant has low levels of intracellular malate under all growth conditions tested. The tpy1 mutant, defective in the transport of pyruvate across the inner membrane, shows repression of gluconeogenesis enzymes under some growth conditions, particularly high levels of ethanol in the medium. These conditions also lead to low levels of malate in the cells. Intracellular levels of malate in these mutants, and in the wild type, are correlated with the levels of gluconeogenic enzymes present. The ability of isolated mutant mitochondria to phosphorylate ADP is shown to be consistent with the interpretation that they are defective in inner membrane transport, although as yet no evidence is available that these defects are the primary lesions in the two mutants. The data are consistent with two general models. In one, the exhaustion of an extramitochondrial corepressor or introduction of a coinducer by mitochondrial activity triggers the induction of gluconeogenic enzyme synthesis. In the second, the mitochondria themselves trigger this induction, but only when the tricarboxylic acid cycle is able to operate at a high level.     

Transformation and recombination in rad mutants of Saccharomyces cerevisiae

Summary Disruption/deletion mutations in genes of the RAD52 epistasis group of Saccharomyces cerevisiae were examined for their effects on recombination between single-and double-stranded circular DNA substrates and chromosomal genes in a transformation assay. In rad50 mutants there was a small reduction in recombination with single-stranded DNA at the leu2-3, 112 allele; in addition there was an almost complete elimination of recombination at trpl-1 for both single- and double-stranded DNA. Reintroduction of a wild-type RAD50 gene on a replicating plasmid carrying CEN4 restored recombinational competence at trpl-1, indicating that rad50 is defective in gene replacement of this allele. In rad52 mutants a reduction of 30%-50% in recombination involving either single- or double-stranded circular DNA was observed in each experiment when compared to the wild type. This reduction of recombination in rad52 mutants was similar for recombination at the ura352 mutant locus where only integration events have been observed, and at the trpl-1 mutant locus, where recombination occurs predominantly by gene replacement. Neither the rad54 nor the rad57 mutations had a significant effect on recombination with single- or double-stranded DNA substrates.     

Decreased mitochondrial biogenesis in temperature-sensitive cell division cycle mutants of Saccharomyces cerevisiae

The temperature-sensitive cell division cycle (cdc) G1 mutants cdc28 and cdc35 show decreased mitochondrial volumes with respect to the wild type strain A364A (WT) at the restrictive temperature. Of the three criteria of mitochondrial biogenesis studied, that is, number of mitochondria per cell, relative area of the cell occupied by mitochondria, or relative area of mitochondria occupied by inner membranes, only the second indicator was significantly lower in cdc mutants than in the WT. The mitochondrial inner membranes development did not compensate for the decrease in the organelles volume. Apparently, the reduced mitochondrial biogenesis was not due to the temperature shift because the relative area of the cell occupied by mitochondria was already significantly lower at 25°C in cdc mutants. The specific fluxes of oxygen consumption confirmed that the respiratory capacity of cdc mutants is largely impaired in respect to the WT. Cdc28 and cdc35 mutants of Saccharomyces cerevisiae had been previously shown to exhibit high respiratory quotients (from 3 to 7) in respect to the WT (RQ 1.0), which correlated with carbon and energy uncoupling probably the result of glucose-induced catabolite repression [Aon MA, Mónaco ME, Cortassa S (1995) Exp Cell Res 217, 42–51; Mónaco ME, Valdecantos PA, Aon MA (1995) Exp Cell Res 217, 52–56].     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae.

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both promitochondria and mitochondria obtained from O2-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N2) to elaidic (O2)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O2-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.     

Molecular dissection of mitotic recombination in the yeast Saccharomyces cerevisiae

Recombination plays a central role in the repair of broken chromosomes in all eukaryotes. We carried out a systematic study of mitotic recombination. Using several assays, we established the chronological sequence of events necessary to repair a single double-strand break. Once a chromosome is broken, yeast cells become immediately committed to recombinational repair. Recombination is completed within an hour and exhibits two kinetic gaps. By using this kinetic framework we also characterized the role played by several proteins in the recombinational process. In the absence of Rad52, the broken chromosome ends, both 5' and 3', are rapidly degraded. This is not due to the inability to recombine, since the 3' single-stranded DNA ends are stable in a strain lacking donor sequences. Rad57 is required for two consecutive strand exchange reactions. Surprisingly, we found that the Srs2 helicase also plays an early positive role in the recombination process.     

Mechanisms and control of meiotic recombination in the yeast Saccharomyces cerevisiae

Recent studies in Saccharomyces cerevisiae have provided new insights in our understanding of the molecular mechanisms of meiotic recombination. Meiosis-specific DNA double-strand breaks have been detected and have been shown to be the lesions that initiate recombination events. These are located mostly in promoter regions where the chromatin is in an open configuration, and cluster in domains along the chromosome. They are likely to be made by a topoisomerase II-like protein encoded by the SPO11 gene. Several DNA intermediates in the meiotic double strand-break repair pathway have been characterised and several multi-protein complexes have been identified and shown to be involved at different steps in the repair pathway. The conservation of these protein complexes in higher eukaryotes suggests that the meiotic recombination mechanism could be conserved. With the application of the well characterised genetical, molecular, cytological and biochemical techniques and the recently developed technology for genomic studies (biochips), we can expect a rapid increase in our comprehension of the meiotic recombination process.     

Binding of yeast killer toxin to a cell wall receptor on sensitive Saccharomyces cerevisiae.

35S-labeled killer toxin protein bound to cells of sensitive Saccharomyces cerevisiae S14a. Strains that were resistant to toxin through mutation in the nuclear genes kre1 kre2 bound toxin only weakly. Non-radioactive toxin competed effectively with 35S-labeled toxin for binding to S14a, but did not compete significantly in the binding to mutant kre1-1. This implied that binding to kre1-1 was nonspecific. A Scatchard analysis of the specific binding to S14a gave a linear plot, with an association constant of 2.9 x 10(6) M-1 and a receptor number of 1.1 x 10(7) per cell. Killer toxin receptors were solubilized from the cell wall by zymolyase digestion. Soluble, non-dialyzable cell wall digest from S14a competed with sensitive yeast cells for 35S-labeled toxin binding and reduced toxin-dependent killing of a sensitive strain. Wall digest from kre1-1 competed only weakly for toxin binding with sensitive cells and caused little reduction of toxin-dependent killing. Although the abundant (1.1 x 10(7) per cell) receptor appeared necessary for toxin action, as few as 2.8 x 10(4) toxin molecules were necessary to kill a sensitive cell of S14a. The kinetics killing of S14a suggested that some component was saturated with toxin at a concentration 50-fold lower than that needed to saturate the wall receptor.