The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway

Abstract

Mitochondrial DNA (mtDNA) is inherited as a protein-DNA complex (the nucleoid). We show that activation of the general amino acid response pathway in rho(+) and rho(-) petite cells results in an increased number of nucleoids without an increase in mtDNA copy number. In rho(-) cells, activation of the general amino acid response pathway results in increased intramolecular recombination between tandemly repeated sequences of rho(-) mtDNA to produce small, circular oligomers that are packaged into individual nucleoids, resulting in an approximately 10-fold increase in nucleoid number. The parsing of mtDNA into nucleoids due to general amino acid control requires Ilv5p, a mitochondrial protein that also functions in branched chain amino acid biosynthesis, and one or more factors required for mtDNA recombination. Two additional proteins known to function in mtDNA recombination, Abf2p and Mgt1p, are also required for parsing mtDNA into a larger number of nucleoids, although expression of these proteins is not under general amino acid control. Increased nucleoid number leads to increased mtDNA transmission, suggesting a mechanism to enhance mtDNA inheritance under amino acid starvation conditions.

Fig. 1. The morphology and distribution of mtDNA nucleoids are regulated by general amino acid control. Cells were grown as indicated, fixed with ethanol and stained with DAPI. (A) Cells of strain ρ^– HS40 grown in YNBD+cas (rich) medium contain a few large, bright DAPI-stained mtDNA nucleoids as indicated by the arrowheads. (B) Cells of strain ρ^– HS40 grown on YNBD (minimal) medium have a greatly increased number of smaller nucleoids. (C) Cells of strain ρ^– HS40 grown in YNBD medium supplemented with isoleucine, leucine and valine (YNBD+ILV) have a few large nucleoids. (D) Cells of strain ρ^– HS40 harboring the plasmid, p238, from which Gcn4p is constitutively expressed, have large numbers of small nucleoids when grown in YNBD medium supplemented with isoleucine, leucine and valine. (E) Cells of strain ρ⁺ 14 WW cultured in YNBD+ILV medium have more and smaller nucleoids than do cells of the petite mutant grown in the same medium. (F) Cells of strain ρ⁺ 14 WW cultured in YNBD medium have an increased number of nucleoids.

Fig. 2. The mtDNA of ρ^– HS40 cells is resolved down to oligomeric sized repeats by activation of the general amino acid control pathway. (A) Diagram of tandemly repeated ρ^– mtDNA undergoing intramolecular recombination to produce circular oligomers. (B) Diagram of the resolution of the various oligomeric states of ρ^– mtDNA by two-dimensional gel electrophoresis. Oligomeric circles, linear DNA and supercoiled molecules are detected as discrete spots by hybridization with a ρ^– HS40-specific probe. (C) and (D) Two-dimensional gel electrophoresis of undigested DNA extracted from cells grown on YNBD medium containing isoleucine, leucine and valine (C) or grown on unsupplemented YNBD medium (D). A 20 μg sample of DNA was resolved by two-dimensional gel electrophoresis and hybridized with a probe specific for ρ^– HS40 mtDNA. As a loading control, 1 μg of total DNA from ρ^– HS40 cells linearized by digestion with EcoRV was run only in the second dimension. The mtDNA repeat of HS40 contains a single EcoRV site so that all of the mtDNA in the loading control is observed as a 0.76 kb band, corresponding to the length of the unit repeat. (E) Relative amount of mtDNA oligomers in 20 μg samples of mtDNA from cells grown in YNBD medium with or without isoleucine, leucine and valine.

Fig. 3. Changes in nucleoid number require ABF2 and MGT1. The number of mtDNA nucleoids was examined by DAPI staining of ρ^– HS40 wild-type (A and B), abf2Δ (C and D), mgt1Δ (E and F), ρ⁺ 14 WW wild-type (G and H) and mgt1Δ (I and J) cells grown in either YNBD medium supplemented with isoleucine, leucine and valine (YNBD+ILV) or in YNBD medium alone, as indicated.

Fig. 4. The abf2Δ and mgt1Δ mutant alleles inhibit the production of small circular oligomers of ρ^– HS40 mtDNA induced by activation of the general amino acid control pathway. One-dimensional gel electrophoresis of ∼20 μg samples of total DNA was used to resolve the different oligomeric forms of mtDNA. YNBD medium was supplemented with isoleucine, leucine and valine (ILV) as indicated in the figure. Supercoiled species are indicated as sc. As a loading control, 1 μg of total DNA was linearized by digestion with EcoRV and detected with an HS40 probe (lower panel).

Fig. 5. Ilv5p is required for the general amino acid control-induced nucleoid redistribution. Wild-type, ilv2Δ and ilv5Δ ρ^– HS40 petite cells transformed with either pRS414 or pRS-gcn4^c, as indicated in the figure, were grown in YNBD+ILV medium and stained with DAPI.

Fig. 6. The production of small, circular oligomers of ρ^– HS40 mtDNA by constitutive expression of Gcn4p is not inhibited in ilv2Δ or ilv5Δ cells. One-dimensional gel electrophoresis of ∼20 μg samples of total DNA was used to resolve the different oligomeric forms of mtDNA found in wild-type, ilv2Δ and ilv5Δ cells grown in YNBD+ILV medium. The cells harbored either an empty vector (pRS416) or a vector expressing the constitutively expressed gcn4^c allele (pRS416-gcn4^c). As a loading control, 1/20 of the DNA sample used in the upper panel was linearized by digestion with EcoRV and detected with an HS40 probe (lower panel).

Fig. 7. The transmission of a neutral ρ^– genome in a cross to a ρ⁺ tester strain is increased by constitutive GCN4 expression. A VAR1 petite strain, containing either the GCN4 (□) or the gcn4^c (•) allele (p164 and p238, respectively), was mated to the ρ⁺ tester for 3 h and the diploid progeny were grown in liquid YNBD+ILV medium (containing the necessary nutritional supplements) before plating. Aliquots of the mating mixture were plated at various time points to select for the diploid progeny, and the fraction of respiratory-incompetent colonies was analyzed by TTC overlay (Ogur et al., 1957).