Lethal Interaction of Nuclear and Mitochondrial Genotypes in Drosophila melanogaster

Abstract

Drosophilamelanogaster, like most animal species, displays considerable genetic variation in both nuclear and mitochondrial DNA (mtDNA). Here we tested whether any of four natural mtDNA variants was able to modify the effect of the phenotypically mild, nuclear tko^25t mutation, affecting mitochondrial protein synthesis. When combined with tko^25t , the mtDNA from wild strain KSA2 produced pupal lethality, accompanied by the presence of melanotic nodules in L3 larvae. KSA2 mtDNA, which carries a substitution at a conserved residue of cytochrome b that is predicted to be involved in subunit interactions within respiratory complex III, conferred drastically decreased respiratory capacity and complex III activity in the tko^25t but not a wild-type nuclear background. The complex III inhibitor antimycin A was able to phenocopy effects of the tko^25t mutation in the KSA2 mtDNA background. This is the first report of a lethal, nuclear-mitochondrial interaction within a metazoan species, representing a paradigm for understanding genetic interactions between nuclear and mitochondrial genotype relevant to human health and disease.

Keywords: cybrid; cytochrome b; melanotic nodules; mtDNA copy number; respiration.

Figure 1

Developmental phenotypes of cybrid strains (A) Egg-to-adult development times of flies of the indicated sex and genotype. Note that the mtKSA2 haplotype was pupal lethal in the ntko^25t background. (B) Late pupal mtDNA copy number, arbitrarily normalized to that of a wild-type ORT adult strain. All data plotted as means ± SD from at least four replicate vials. Asterisks (*, **, ***) denote statistically significant differences between nuclear backgrounds, for flies of a given sex and mtDNA haplotype (Student’s t-test, P < 0.05, 0.01, 0.001, respectively).

Figure 2

Hemolymph abnormalities in cybrid lines (A) Images of ntko^25t mtKSA2 flies during wandering L3 and pupal stage. Red ellipses (left-hand panel) are drawn around an example of melanotic nodules. After dissection (right-hand panel), most nodules were observed to be free of association with any specific tissue. (B) Total hemocyte counts from hemolymph of individual L3 larvae from indicated cybrids (means ± SD of at least 15 larvae of each genotype). Results of ANOVA followed by Tukey post hoc HSD test indicated by horizontal lines joining values showing significant differences (95% CI, *, *** denoting P < 0.05 or < 0.001 respectively).

Figure 3

Eclosion frequency of nORT cybrids cultured on medium containing antimycin A (A) Egg-to-adult eclosion frequency (i.e., percentage of eggs completing development and emerging as adults) of the indicated cybrid lines, cultured in 5 μg/ml antimycin A. Note that all emerging flies in a given culture were of the same genotype. Means ± SEM of 7 or 8 replicate vials (∼65 eggs laid per vial). Significant differences based on one-way ANOVA with post-hoc Tukey HSD test. The dose of antimycin was based on preliminary trials to confirm the sub-lethal concentration inferred from the literature (Frei et al. 2005). (B) Egg-to-adult eclosion frequency for progeny flies of each genotype indicated, in the cross tko^25t / FM7 x tko^25t / Y, cultured on medium containing 5 μg/ml antimycin A. The flies used in the cross were all in the control mtDNA background mtWT5A. Note that males were more severely affected by the drug than females (which was observed for all genotypes tested), but the proportion of flies of a given sex eclosing on antimycin A was independent of tko genotype (chi-squared test, P > 0.05 for both sexes). Data are from a single, large-scale experiment (n = 940) of sufficient size to generate statistically robust values.

Figure 4

Respirometry of homogenates from cybrid strains State 3 respiration on cI-, cIII- and cIV-linked substrate mixes, of homogenates from pupae of the sex and genotypes indicated. Asterisks above the bars (*, **, ***) indicate significant differences between the nuclear backgrounds (Student’s t-test, P < 0.05, 0.01, 0.001, respectively); filled circles (•, ••, •••) denote significant differences between mtDNA haplotypes for a given nuclear background (Bonferroni-corrected Student’s t-test, P < 0.05, 0.01, 0.001, respectively; ns – not significant).

Figure 5

Respiratory activities in pupal homogenates of cybrid strains (A, B) Blue-native electrophoresis and in-gel histochemistry for (A) cI, (B) cIV, of strains of the indicated mtDNA haplotypes in the nORT and ntko^25t backgrounds as shown. (C) cIII assay (means ± SD of four biological replicates) from strains containing the indicated mtDNAs in the nORT and ntko^25t backgrounds as shown. ** denotes statistically significant differences between the nuclear backgrounds for a given mtDNA haplotype (Student’s t-test, P < 0.01).

Figure 6

Molecular modeling and sequence alignments (A) Crystal structure of bovine cIII, showing the monomer, dimer and subunit interface between cytochrome b (mtCYTB, dark blue) and core 1 (UQCRC1, light blue). Other subunits shown in green. (B) Details of the structure, showing conserved residue D20 of mtCYTB, and its proximity contacts with other amino acids. In the bovine structure, D20 of mtCTYB interacts electrostatically with R436 of UQCRC1 and with K6 of mtCYTB. In Drosophila, K6 is replaced by N7, which is still able to interact with D21 (equivalent to bovine D20) via a hydrogen bond, whereas the charged groups of D21 and R460 (replacing bovine R436) should interact electrostatically. The D21N replacement would abolish this electrostatic interaction (the amino groups of N21 and R460 may even repel), weakening the overall structure, and potentially interfering with the complex reaction mechanism of the enzyme. (C) Partial amino acid alignment of the cytochrome b polypeptides of the four species, indicated (Bovine – Bos taurus, NCBI AAZ95348, commencing with M1; Human – Homo sapiens, NCBI ADT79912; Drosophila – D. melanogaster, NCBI AAA69714; Yeast – Saccharomyces cerevisiae, NCBI CAA24073. The conserved aspartate that is replaced by an asparagine in mtKSA2 is shown against yellow background. (D) Partial sequence of UQCR-C1 polypeptide of D. melanogaster (Refseq – NCBI Q9VFF0, commencing at R405; inferred sequences for strains ORT and tko^25t, based on DNA sequencing). The conserved arginine involved in interactions with cytochrome b is shown against turquoise background.