An oligomycin-resistant adenosine triphosphatase and its effects on cellular growth, mitochondrial oxidative phosphorylation and respiratory proton translocation in Saccharomyces cerevisiae.

Mutations at the OLI 1 or OLI 2 loci of mitochondria DNA in Saccharomyces cerevisiae are associated with a diminished growth rate in nutritionally suboptimal cultures supplemented with an oxidizable carbon source. In the case of mutant OR146(OLI1) there is a 35% loss of mitochondrial protein during fractionation in vitro, suggesting that the mutationally altered adenosine triphosphatase(ATPase) confers some instability on the mitochondrial membrane. The possibility is discussed that this reflects an unstable mitchondrial population in vivo, leading the observed growth deficiency. Mitochondria from mutant OR146 at the OLI 1 locus show a relatively oligomycin-resistant State-3 respiration, but the same ADP/O and respiratory-control quotients as the isonuclear wild-type. A slightly lowered Qo2 with NADH-linked substrates was observed and is discussed. For both strains the apparent H+/O ratios were close to 4 with pyruvate, ethanol and alpha-oxoglutarate, but consistently lower with succinate and citrate. For each substrate a characteristic t 1/2 (time for half-decay of the transmembrane pH differential) range was found, consistent with the view that the substrates effecitvely carry the protons back across the membrane. As expected, H+/O ratios were independent of t 1/2 for all substrates, with the exception of alpha-oxoglutarate in the case of the wild-type, where an inverse correlation was found. The lack of this correlation in the case of the mutant was the only apparent difference in the translocation parameters observed. A hypothesis relating this to the functioning of the oligomycin-resistant ATPase is proposed.     

Biogenesis of mitochondria 36

Summary The isolation and characterisation of a mutant affecting the assembly of mitochondrial ATPase is reported. The mutation confers resistance to oligomycin and venturicidin and sensitivity of growth on nonfermentable substrates to low temperature (19°). Genetic analysis indicates that the phenotype is due to a single mutation located on the mitochondrial DNA which is probably allelic with the independently isolated oligomycin resistance mutation [oli1-r].Growth of the mutant at the non-restrictive temperature (28°) yields mitochondria in which the ATPase appears more sensitive to oligomycin than that of the sensitive parental strain. However, when the enzyme is isolated free from the influence of the membrane strong resistance to oligomycin is evident. These data suggest that the component responsible for the oligomycin resistance of the ATPase is part of or subject to interaction with the mitochondrial inner membrane.Measurements of the ATPase content of mitochondria indicate that ATPase production is impaired during growth at 19° C. In addition, studies of the maximum inhibition of mitochondrial ATPase activity by high concentrations of oligomycin suggest a selective lesion in ATPase assembly at low temperature. The nett result is that during growth at 19° only about 10% of the normal level of ATPase is produced of which less than half is membrane integrated and thus capable of oxidative energy production.We propose that the mutation affects a mitochondrially synthesised membrane sector peptide of the ATPase which defines the interaction of F₁ ATPase with specific environments on the mitochondrial inner membrane.     

Nucleo-cytoplasmic interaction between oligomycin-resistant mutations in Saccharomyces cerevisiae

1.A single-gene nuclear mutant of Saccharomyces cerevisiae, isolated as oligomycin-resistant, exhibits in vivo cross-resistance to venturicidin and collateral sensitivity to Synthalin. All three compounds are inhibitors of mitochondrial oxidative phosphorylation. Oligomycin resistance and Synthalin sensitivity are recessive, while venturicidin resistance is dominant. 2. Acytoplasmic mutant, also isolated as oligomycin-resistant, shows collateral sensitivity to both Synthalin and venturicidin. All three traits undergo mitotic segregation in diploids formed by crossing mutant and normal halpoids. 3. A novel nucleocytoplasmic interaction is observed in diploids formed by crossing haploid strains containing the nuclear and the cytoplasmic mutations, respectively. The dominant venturicidin resistance determined by the nuclear gene undergoes mitotic segregation, which results from a suppression of the nuclear phenotype by the cytoplasmic mutation. When a diploid mitotic segregant contains primarily mutant-type mitochondria, venturicidin resistance is completely suppressed. In haploids containing both the nuclear and cytoplasmic mutations, suppression is only partial. 4. Oxidative phosphorylation and ATPase in mitochondrial fractions isolated fromcytoplasmic mutant cells are less sensitive to inhibition by oligomycin than normal, but in vitro sensitivity to venturicidin is not significantly changed. In similar mitochondrial fractions isolated from normal and nuclear mutant cells, no significant differences in sensitivity to either inhibitor are detected. 5. The molecular basis for the nucleocytoplasmic suppression of venturicidin resistance may involve participation of mitochondrial membrane, plasma membrane or both. Either mitochondria can undergo changes in venturicidin sensitivity upon isolation, or the molecular entity which controls access of venturicidin to the mitochondria resides outside of the organelles. 6. Our data establish that aspects of the response in vivo of both venturicidin and Snythalin are controlled by the mitochondrial genome. 7. The nucleocytoplasmic interaction described here is the first example in which a specific restricted mitochondrial mutation modifies the phenotypic expression of a nuclear gene.     

Involvement of IS1 in the dissociation of the r-determinant and RTF components of the plasmid R100.1

Summary Detailed mapping localized the PHO 1 mutation between the OLI 2 and OLI 4 loci on mitochondrial DNA of Saccharomyces cerevisiae.In its mitochondrially integrated form, the PHO 1-ATPase³ was difficult to identify either immunologically or by specific inhibitors like oligomycin and DCCD. Solubilization by Triton X-100 allowed unambiguuous identification of this enzyme as an authentic mitochondrial ATPase. However, Triton extraction produced a 2 to 3 fold enhancement of the PHO 1-ATPase activity which also became drastically cold-sensitive. The wild type ATPase was neither activated nor made cold-labile by solubilization, and retained full sensitivity to oligomycin and DCCD.Sucrose gradient analysis of the Triton-extracted ATPase from wild type, PHO 1 mutant and rho ^- strains showed a density difference between the solubilized PHO 1-and wild type ATPase, and similarity between solubilized PHO 1-and rho ^- ATPase (F₁).Whole cells of the PHO 1 mutant present considerably increased respiration rates.Comparison of oligomycin-sensitivity in whole cells, coupled isolated mitochondria and membrane-bound ATPase indicates a contrast between oligomycin-resistance of the ATPase and oligomycin-sensitivity of in vivo or in vitro coupling systems, which might characterize the products of this region of mitochondrial DNA.     

An oligomycin-resistant Mg2+-dependent ATPase in rabbit bone marrow mitochondria

Summary Rabbit bone marrow mitochondria isolated by differential centrifugation showed typical oxypolarographic tracings with glutamate oxidation with ADP:O ratio of 2.9. Similar results were obtained with liver mitochondria of the same animal. When marrow mitochondria were oxydizing a substrate such as glutamate, added MgCl₂ markedly stimulated state-4 respiration giving a respiratory rate identical to that of state-3. In contrast, no Mg²⁺-stimulation was observed with liver mitochondria. Oligomycin completely blocked the stimulation by Mg²⁺ but further addition of 2,4-dinitrophenol reactivated the oxygen consumption by uncoupling. Further purification of marrow mitochondria by density gradient centrifugation in Percoll provided identical oxypolarographic results. Moreover, when marrow mitochondria were incubated without Mg²⁺, they showed a low ATPase activity that was stimulated by 2,4-dinitrophenol and blocked by oligomycin. The presence of Mg²⁺ in the incubation medium uncovered an additional ATPase activity which was resistant to oligomycin and apparently unaffected by 2,4-dinitrophenol. It is concluded that bone marrow mitochondria possess two types of ATPase activity distinguished on the basis of their reactivity with oligomycin, 2,4-dinitrophenol and Mg²⁺.Abbreviations EDTA ethylenediamine tetraacetate - DNP 2,4-dinitrophenol - BSA bovine serum albumin - BMM bone marrow mitochondria - LM liver mitochondria - Oligo. oligomycin - Anti A antimycin A Howard Hughes Investigator.     

Mitochondrial Genetics. Xii. an Oligomycin-Resistant Mutant Localized at a New Mitochondrial Locus in SACCHAROMYCES CEREVISIAE

Three antibiotic-resistance mutations were isolated from strain FL496–2B: two are independent Mendelian genes, one conferring both oligomycin and venturicidin resistance (oli^R₄₉₆) and the other conferring cycloheximide resistance (cyh^R₄₉₆). The third is a mitochondrial mutation, O^R₉, and confers a low level of oligomycin resistance to cells (in vivo) but not to the extracted mitochondrial ATPase (in vitro). This mutation is located on the mitochondrial DNA at a new locus [OLI4] linked to [OLI2] and independent from [OLI1] and [OLI3] and from the other mitochondrial loci.

All three mutations (O ^R₉, oli^R₄₉₆, cyh^R₄₉₆ ) were found without any selection, in the same prototrophic haploid strain, which contained unknown resistances to antibiotics.

Some physiological, genetical and biochemical properties of the mitochondrial mutation are described.

     

Extrachromosomal oligomycin-resistant mutants of the petite-negative yeast Kluyveromyces lactis. Properties of mitochondrial ATPase and cross-resistance to inhibitors of phosphoryl transfer reactions

Summary The mitochondrial ATPase from oligomycin-resistant mutants which map on different regions of an extrachromosomal DNA (01 and 011 class mutants) showed an increased resistance to oligomycin and venturicidin when assayed in vitro as compared to the sensitive strains.The resistance to oligomycin of the isolated mitochondrial ATPase from 01 class mutants was higher than that of the 011 class mutants.Cross resistance of the oligomycin-resistant mutants to the antibiotics peliomycin and ossamycin, which also inhibit phosphoryl transfer reactions in mitochondria (Walter et al., 1967), was observed, 01 mutants being more resistant to ossamycin than 011 class mutants. At the concentrations of peliomycin studied, no difference in sensitivity among both groups of oligomycin-resistant mutants could be detected.Mitochondrial respiration and isolated mitochondrial ATPase activity are sensitive to venturicidin, suggesting that the previously observed (Brunner et al., 1977) in vivo venturicidin resistance of K. lactis is probably due to an impairment of the influx of the drug at the level of the plasma membrane.     

Biogenesis of mitochondria 23. The biochemical and genetic characteristics of two different oligomycin resistant mutants ofSaccharomyces cerevisiae under the influence of cytoplasmic genetic modification

Two classes ofSaccharomyces cerevisiae mutants resistant to oligomycin, an inhibitor of mitochondrial membrane bound ATPase are described. Biochemical analysis shows thatin vitro the mitochondrial ATPase of both types of mutant are sensitive to oligomycin.In vivo sensitivity of the mutants to oligomycin can be demonstrated following anaerobic growth of the cells, which grossly alters the mitochondrial membrane and renders the ATPase of the mutants sensitive to oligomycin. It is concluded that the mutation to oligomycin resistance in both mutant types is phenotypically expressed as a change in the mitochondrial membrane. The intact mitochondrial membrane in the wild type cell is freely permeable to oligomycin, whereas the resistant mutant is impermeable to oligomycin; alteration of the mitochondrial membrane during isolation of the organelle or physiological modification of the membranes of the mitochondria by anaerobic growth renders the membranes permeable.These mitochondrial membrane mutants differ in their cross-reference patterns and their genetics. One is resistant to oligomycin only, and behaves like previously reported cytoplasmic mutants. The other shows cross-resistance to inhibitors of mitochondrial protein synthesis as well as to oligomycin; although the mutant appears to arise from a single step mutation its genetic properties are complex and show part-nuclear and part-cytoplasmic characteristics. The implications of the observations are discussed.     

Transfer of cytoplasmic organelles from an oligomycin-resistant Nicotiana cell suspension into tobacco protoplasts yielding oligomycin-resistant cybrid plants

Summary Oligomycin-resistant lines were derived from a Nicotiana sylvestris cell suspension, after N-nitroso-N-methylurea mutagenesis followed by selection in the presence of 0.4 g/ml oligomycin, a specific mitochondrial ATPase inhibitor. One of the lines, oli ^R38 was further analyzed to investigate the role of mitochondria in this resistance. The oli ^R38 line proved to be also highly resistant to venturicidin, another specific inhibitor of mitochondrial ATPase. By the donor-recipient protoplast-fusion procedure the cytoplasmic organelles of oli ^R38 were transferred to protoplasts of Line 92, a line of tobacco plants which contain the cytoplasmic organelles of N. undulata. Cell suspensions prepared from several cybrid plants, containing the cytoplasmic organelles of oli ^R38, exhibited the same level of oligomycin resistance as the oli ^R38 line.     

Mitochondrial oligomycin-resistance mutations affecting the proteolipid subunit of the mitochondrial adenosine triphosphatase.

Two independently isolated oligomycin resistant mutants of Saccharomyces cerevisiae have been studied. The oligomycin resistance is conferred in each case by a single mutation at an oliA locus. In both strains the proteolipid subunit of the mitochondrial ATPase (subunit 9) shows an apparent increase in molecular weight as judged by its mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Variable effects are seen on other subunits. These results suggest that oliA loci may play some role in the determination of proteolipid ATPase subunit.     

Mitochondrial DNA of two independent oligomycin-resistant Chinese hamster ovary cell lines contains a single nucleotide change in the ATPase 6 gene

We have determined the nucleotide sequence of the URF A6L and ATPase 6 genes of the mitochondrial DNA of wild-type Chinese hamster ovary (CHO) cells and of two independently isolated, cytoplasmically inherited CHO mutant cell lines that are resistant to oligomycin, an inhibitor of the mitochondrial ATP synthase (ATPase) complex. Comparison of the nucleotide sequences of the mutants with that of their parental cell line revealed a single nucleotide difference, a G-to-A transition at nucleotide 433 of the ATPase 6 gene. This single base pair change predicts a nonconservative amino acid change, with a glutamic acid residue being replaced by a lysine residue at amino acid 145 of the ATPase 6 gene product in the mutants. This glutamic acid residue and several others in the surrounding amino acid sequence are conserved among all species examined to date. Analyses of several of the biochemical properties of the oligomycin-resistant CHO mutants indicate that the glutamic acid residue at position 145 of subunit 6 of the mitochondrial ATP synthase complex is important for the binding of oligomycin to the enzyme complex, but is not essential for proton translocation.     

Localization on mitochondrial DNA of mutations leading to a loss of rutamycin-sensitive adenosine triphosphatase.

Four cytoplasmic mutants of Saccharomyces cerevisiae showing loss of mitochondrial rutamycin-sensitive ATPase activity but having significant cytochrome oxidase and NADH-cytochrome c reductase have been isolated. Genetic studies indicate the mutations to be closely linked to each other and have been assigned to a new locus, PHO1. The mutations show a low frequency of recombination with the OL12 locus, suggesting a linkage to this marker. They are not, however, linked to the OLI1 locus. Linkage of the ATPase mutations to the OLI2 locus is also indicated by restoration of wild-type diploids by sigma- clones that retain the segment of mitochondrial DNA carrying OLI2. Based on the recombinants issued from crosses of the mutants with a triple drug-resistant strain and an analysis of the resistance markers present in sigma- clones that are effective in restoring a wild-type phenotype, the PHO1 locus has been placed in the segment of DNA located between PAR1 and OLI2.     

Oligomycin-resistant mitochondrial ATPase from mouse fibroblasts.

Fourteen oligomycin-resistant LM(TK-) clones were isolated following the mutagenesis of minicells. In the absence of oligomycin, the mutants grew with population doubling times similar to that of the wild type (1 day). In 3 or 5 microgram oligomycin/ml the doubling times of the mutants were 1.2-2.5 days. Both stable and unstable classes were represented among the oligomycin-resistant mutants. Mitochondrial ATPase activities of the mutants were 1.3-1130 times more resistant to oligomycin than the wild type. The mitochondrial ATPase of OLI 14 was found to be bound firmly to the mitochondrial membrane, showed no alteration in the pH optimum compared to wild-type, and exhibited increased resistance to DCCD and venturicidin. These results are consistent with the conclusion that oligomycin resistance in these mutants results from altered mitochondrial ATPase.     

Nuclear and mitochondrial revertants of a mitochondrial mutant with a defect in the ATP synthetase complex

Summary Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20°C). At 28°C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20°C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.Abbreviations DCCD dicyclohexylcarbodiimide - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - SMP submitochondrial particles - mit^- mitochondrial point mutant     

A novel mitochondrial DnaJ/Hsp40 family protein BIL2 promotes plant growth and resistance against environmental stress in brassinosteroid signaling

Plant steroid hormones, brassinosteroids, are essential for growth, development and responses to environmental stresses in plants. Although BR signaling proteins are localized in many organelles, i.e., the plasma membrane, nuclei, endoplasmic reticulum and vacuole, the details regarding the BR signaling pathway from perception at the cellular membrane receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) to nuclear events include several steps. Brz (Brz220) is a specific inhibitor of BR biosynthesis. In this study, we used Brz-mediated chemical genetics to identify Brz-insensitive-long hypocotyls 2-1D (bil2-1D). The BIL2 gene encodes a mitochondrial-localized DnaJ/Heat shock protein 40 (DnaJ/Hsp40) family, which is involved in protein folding. BIL2-overexpression plants (BIL2-OX) showed cell elongation under Brz treatment, increasing the growth of plant inflorescence and roots, the regulation of BR-responsive gene expression and suppression against the dwarfed BRI1-deficient mutant. BIL2-OX also showed resistance against the mitochondrial ATPase inhibitor oligomycin and higher levels of exogenous ATP compared with wild-type plants. BIL2 participates in resistance against salinity stress and strong light stress. Our results indicate that BIL2 induces cell elongation during BR signaling through the promotion of ATP synthesis in mitochondria.     

Studies on energy-linked reactions: isolation and properties of mitochondrial venturicidin-resistant mutants of Saccharomyces cerevisiae.

Venturicidin is a specific inhibitor of aerobic growth of yeast and has no effect on fermentative growth, a result which is consistent with its known mode of action on mitochondrial oxidative phosphorylation. Venturicidin-resistant mutants of Saccharomyces cerevisiae have been isolated and form two general classes: class 1, nuclear mutants which are resistant to a variety of mitochondrial inhibitors and uncouplers, and class 2, mitochondrial mutants of phenotype VENR OLYR and VENR TETR in vivo. VENR OLYR mutants show a high degree of resistance to venturicidin and oligomycin at the whole cell and mitochondrial ATPase level but, in contrast, no resistance at the mitochondrial level is observed with VENR TETR mutants. Venturicidin resistance/sensitivity can be correlated with two binding sites on mitochondrial ATPase, one of which is common to the oligomycin binding site and the other is common to the triethyl tin binding site. Biochemical genetic studies indicate that two mitochondrial genes specify venturicidin resistance/sensitivity and that the mitochondrial gene products are components of the mitochondrial ATPase complex.     

Effects of oligomycins on adenosine triphosphatase activity of mitochondria isolated from the yeasts Saccharomyces cerevisiae and Schwanniomyces castellii

Functional mitochondria with respiratory control were isolated from the yeasts Saccharomyces cerevisiae and Schwanniomyces castellii. The presence of site I in Schw. castellii was indicated by higher ADP/O ratio than in S. cerevisiae where this site is absent. The ATPase Vmax was higher in S. cerevisiae than in Schw. castellii mitochondria. The latter was increased by the DR12 nuclear mutation. Nevertheless, the stimulation by heat and the inhibition profile of oligomycins on mitochondrial F1-F0 ATPase activities were similar in all three tested strains. In S. cerevisiae and Schw. castelli wild type or mutant mitochondria, the well-known inhibition of F1-F0 ATPase activity by low concentrations of oligomycins is abolished at high inhibitor concentrations near 60microg/ml suggesting uncoupling of F1 activity. At still higher oligomycin concentration the ATPase activity of both species and mutant is again strongly inhibited, suggesting an inhibitory effect on yeast F1 activity not detected so far.     

Genetics of oxidative phosphorylation: mitochondrial loci determining ossamycin-, venturicidin- and oligomycin-resistance in yeast

Summary With a view towards identifying new ATPase loci on the mitochondrial genome a large number of oligomycin-, ossamycin- and venturicidin-resistant mutants were isolated after MnCl₂ mutagenesis. The mutants were subjected to mass-screens which divided them into different cross-resistance phenotype-classes and also distinguished the common OLI1 mutations from the mutations at all other loci.Allelism tests between examples of the different classes of phenotype indicated that the majority of mutations in the population mapped at the previously known loci OLI1, OLI2, OLI3 and OLI4. Mutations conferring specific ossamycin resistance defined two new loci, namely OSS1 and OSS2 which are linked to the OLI2 and OLI1 loci respectively. A few rare mutations comprise a new locus OLI5 which is linked to the OLI1 locus (12.6% total recombination).In conclusion we can now say that there are two unlinked segments of the mitochondrial genome, each of which is composed of several distinct, genetically-linked loci. One segment contains the OLI1, OLI3, OLI5 and OSS2 loci and the other the OLI2, OLI4 and OSS1 loci. The phenotypically-distinguishable mutations described herein should facilitate fine-structure mapping of these two segments.     

Nuclear-extranuclear interactions affecting oligomycin resistance in Aspergillus nidulans.

Summary The extranuclear mitochondrial oligomycin-resistant mutation ofAspergillus nidulans, (oliA1), was transferred asexually into four nuclear oligomycin-resistant strains of different phenotypes. In all four cases, the possession of the nuclear plus extranuclear mutation led to an increase in the in vivo level of oligomycin resistance. In two cases, the altered cytochrome spectrum and impaired growth ability determined by (oliA1) were suppressed by the nuclear mutations. In the third case, the in vitro oligomycin resistance of the double mutant ATPase was dramatically increased above that of either of the component single mutant strains, indicating a synergystic interaction between the nuclear and extranuclear gene products. In the fourth case, the double mutant became cold-sensitive.A new extranuclear mitochondrial oligomycin-resistant mutation (oliB332) is described. This mutant is phenotypically similar to, though not identical with, (oliA1) but is separable by recombination.A range of nuclear oligomycin-resistant mutants have been mapped. Despite presenting five distinctly different phenotypes, they all map at the same locus.     

Genetic Analysis of Multiple Drug Cross Resistance in SACCHAROMYCES CEREVISIAE: a Nuclear-Mitochondrial Gene Interaction

A mutant of the yeast Saccharomyces cerevisiae, cross resistant to several antibiotics, was isolated in our laboratory and subjected to genetic analysis. Tetrad analysis of diploids obtained from crosses between the resistant mutant and a sensitive wild-type strain suggest that the multiple resistance to the five agents, oligomycin (OLI), rhodamine 6G (RHG), tetracycline (TCN), chloramphenicol (CAP) and cycloheximide (CHX) is determined by a single nuclear gene, ant1, and requires several cytoplasmic genes for expression of resistance to oligomycin, rhodamine 6G and tetracycline. --Vegetatively growing diploid clones derived from the cross ant1 [RHO+] X +[RHO+] show mitotic segregation of two phenotypic classes for the drugs OLI, RHG TCN. Diploids derived from the two reciprocal crosses, ant1 [RHO+] X +[RHO-] and ant1 [RHO-] X +[RHO+], fail to exhibit mitotic segregation. These results are consistent with our hypothesis concerning the involvement of cytoplasmic loci. They suggest, in addition, that these loci are associated with mitochondrial DNA (mtDNA). --Evidence for this association is provided by the demonstration of genetic linkage between the cytoplasmic loci involved in the interaction, RHG-1, TCN-1 and OLI-5, and two well-characterized mitochondrial loci, ERY and CAP. --We have mapped the nuclear ant1 locus 3.3 cM from the centromere-linked gene, leu1, on the same side of the centromere of chromosome VII as leu1. --In the light of these findings, we discuss the claims made by several authors of the episomal nature of mutations similar to the one described here, as well as of the possible involvement of yeast 2 mu DNA in such mutations.