Characterization of epitopes of the yeast mitochondrial H+-ATPase complex recognized by monoclonal antibodies

Nine monoclonal antibodies which react with the beta subunit of the yeast mitochondrial H+-ATPase and three which react with a 25 kDa subunit of the enzyme complex (P25) have been characterized. Competitive binding studies indicated the presence of at least four antigenic regions on the beta subunit of the enzyme complex. One antigenic region of the beta subunit is recognized by two monoclonal antibodies RH 57.1 and RH 45.5 which inhibit the ATPase activity to different degrees. Antibody RH 48.6 appears to bind to a second region on the beta subunit and has no effect on the ATPase activity. A third region of the beta subunit is recognized by antibodies RH 51.4 and RH 72.1. RH 51.4 has no effect on the ATPase activity, whereas RH 72.1 stimulates ATPase activity. Antibody RH 32.4 which has no effect on the ATPase activity appears to bind to the fourth epitope of the beta subunit. All three monoclonal anti-P25 antibodies, RH 66.3, RH 41.2 and RH 37.0, apparently bind to the same antigenic region on this subunit. Two of the monoclonal anti-beta antibodies RH 48.6 and RH 51.4 were found to be very effective in immunoprecipitating the whole H+-ATPase complex in a solid phase system. However, the other monoclonal antibodies (and also a polyclonal antiserum) appear to induce the dissociation of one or more of the H+-ATPase subunits by their binding to the epitopes on the beta or the P25 subunits.     

mit-Mutations in the structural gene of subunit III of cytochrome oxidase in Saccharomyces cerevisiae

Two-dimensional electrophoretic analysis of the mitochondrial translation products of four mit-mutants indicate that subunit III of cytochrome oxidase is the only mitochondrial translation product affected by mutations in the oxi2 region of the mtDNA. Mitochondria of two of these mutants synthesize new products which coprecipitate with an anticytochrome oxidase antiserum and produce proteolytic digests similar to those of subunit III of the enzyme complex. These data strongly support the suggestion that the oxi2 region of the yeast mtDNA contains the structural gene of subunit III of cytochrome oxidase.     

Mitochondrial H+-ATPase in mutants of Saccharomyces cerevisiae with defective subunit 8 of the enzyme complex

Mutants of Saccharomyces cerevisiae carrying defined lesions in the mitochondrial aap1 gene, coding for membrane subunit 8 of the H+-ATPase, have been investigated to examine the consequence of the mutations on the function and assembly of the enzyme complex. These include three mit- mutants, which cannot grow by oxidative metabolism due to their inability to synthesize full-length subunit 8, and three partial revertants of one of the mutants. The mutations in these strains have been previously characterized by DNA sequencing. The use of a monoclonal antibody to the beta subunit of the H+-ATPase as a probe of assembly defect revealed that the presence of subunit 8 is essential for the assembly of subunit 6 to the enzyme complex. Mitochondria isolated from the mit- mutants have negligible [32Pi]ATP exchange activity and they exhibited ATPase activity which is not sensitive to inhibition by oligomycin, indicating a defective membrane F0 sector. Normal assembly of subunit 8 (and subunit 6) was observed in the revertant strains, despite 8-9 amino-acid substitutions in the membrane-spanning region of the H+-ATPase subunit 8 in two of the strains. The assembled complex, however, exhibited reduced [32Pi]ATP exchange activity and low sensitivity to oligomycin, indicating that the product of the aap1 gene is a functional subunit of the mitochondrial H+-ATPase.     

Biogenesis of mitochondria. Defective assembly of the proteolipid into the mitochondrial adenosine triphosphatase complex in an oli2 mit− mutant of Saccharomyces cerevisiae

A single mutation in the oli2 region of the mitochondrial DNA causes a charge alteration in a mitochondrially translated subunit of the mitochondrial ATPase (subunit 6; apparent M_r 20 000; apparent pI 6.9 and 7.1). This alteration leads to the defective assembly of the proteolipid subunit into the enzyme complex. The mutant, which is able to grow only very slowly by oxidative metabolism at 28°C offers new possibilities for studying the assembly of the membrane sector (F₀) into the mitochondrial ATPase complex and the role of subunit 6 in this process.     

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.     

Biogenesis of mitochondria: Defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H⁺-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H⁺-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized -subunit of the H⁺-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H⁺-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.This paper is No. 61 in the seriesBiogenesis of Mitochondria. For paper No. 60, see Novitskiet al. (1984).     

Evaluation of Pesticide EVects on Arthropod Predator Populations in Soya Bean in Farmers' Fields

The impact of two insecticides applied at locally practised frequencies on the population fluctuations of soya bean arthropods was studied at two farmers' field sites in Indonesia. Temporal recovery was determined from observations taken consecutively at short intervals. Spatial recovery was determined from observations taken at diVerent distances from the unsprayed surroundings. Monocrotophos suppressed predaceous ants, spiders and beetles, but did not reduce all phytophages. Lambda-cyhalothrin had a large impact on generalist predators and suppressed phytophages. Multiple regression analysis showed that temporal recovery was low for generalist predators and highest for lepidopterous larvae, indicating a potential to resurge after spraying. Spatial recover was observed for spiders, beetles, crickets and lepidopterous larvae (Galerucinae and Empoasca sp.), indicating that the presence of refuge areas may encourage recovery. To reduce the dependency on insecticides, farmers require training in phytophage-predator-insecticide interactions through field exercises.     

Length variations in the COII-tRNA(Lys) intergenic region of mitochondrial DNA in Indonesian populations.

The prevalence of a 9-base-pair (bp) deletion between the mitochondrial cytochrome oxidase II (MTCOX*2) and lysine tRNA (MTTK) genes (region V) has been used to estimate the genetic relationships among Asian and Pacific populations. Many East Asian and Pacific Island populations have been examined previously, but the mitochondrial DNA (mtDNA) diversity of the intervening Indonesian archipelago has not previously been systematically examined. The 17,500 islands of Indonesia currently contain nearly 213 million people and extensive cultural, linguistic, and, presumably, genetic diversity. This study of 1091 individuals representing 15 ethnic groups is the most extensive mtDNA survey to date of the Indonesian archipelago. Six distinct length polymorphisms in region V were observed within these 15 populations. The 9-bp deletion was found in every population examined at frequencies comparable to those of previously examined East Asian populations and substantially lower than those in most Pacific Island populations. Despite the inclusion of Austronesian-speaking populations and a Papuan-speaking population, there was no statistically significant heterogeneity in the frequency of the 9-bp deletion among the 15 populations (p = 0.09). These data indicate that substantial gene flow occurred among the populations at some time in the past. Our observations of no significant correlations between genetic and geographic distances (r = -0.04, p = 0.53) coupled with the extensive cultural and linguistic differences currently within the archipelago suggest that little gene flow among neighboring populations has occurred recently.