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. 2019 Feb 18;8(2):bio041244.
doi: 10.1242/bio.041244.

Gcn5 histone acetyltransferase is present in the mitoplasts

Arianna Montanari  1   2 , Manuela Leo  3 , Veronica De Luca  3 , Patrizia Filetici  4 , Silvia Francisci  3
Affiliations

Gcn5 histone acetyltransferase is present in the mitoplasts

Arianna Montanari et al. Biol Open. .

Abstract

In Saccharomyces cerevisiae the Lysine-acetyltransferase Gcn5 (KAT2) is part of the SAGA complex and is responsible for histone acetylation widely or at specific lysines. In this paper we report that G CN5 deletion differently affects the growth of two strains. The defective mitochondrial phenotype is related to a marked decrease in mtDNA content, which also involves the deletion of specific regions of the molecule. We also show that in wild-type mitochondria the Gcn5 protein is present in the mitoplasts, suggesting a new mitochondrial function independent from the SAGA complex and possibly a new function for this protein connecting epigenetics and metabolism.

Keywords: Lysine-acetyltransferase Gcn5; Mitochondria; Mitochondrial DNA; Respiration; Yeast.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Deletion of GCN5 differently affects fermentative and respirative growth. (A,B) Serial dilutions of two WT [W303-1A (A) and D273-10B/A1 (B)] and their derivate gcn5Δ strains grown overnight in YP 2% glucose containing medium were spotted on YP 2% glucose or 3% glycerol containing media and incubated at 28 or 37°C.
Fig. 2.
Fig. 2.
Cross experiment demonstrates the relationship between mtDNA and Gcn5. To obtain diploid competent for respiration it is required that the mtDNA does not derive from gcn5Δ strain. (A,B) Schematic procedure of the two crosses to reintroduce GCN5 gene in the deleted strain. Nucleus with wild-type chromosomal DNA is grey whereas in the presence of GCN5 deletion it is black. MtDNA is shown as small beads; GLY+ or − indicate the capability to grow in glycerol containing medium. (C,D) Serial dilutions of parental strains and diploids of the crosses described in A and B, respectively. (E) Serial dilutions of WT, gcn5Δ and spore W303.2b (obtained from the cross described in A) in which the wild-type phenotype is completely restored. All strains are spotted on YP 2% glucose or in 3% glycerol containing media and incubated at 28 or 37°C.
Fig. 3.
Fig. 3.
Different effects of GCN5 deletion in the strains W303-1A and D273-10B/A1. (A) Fluorescence microscopy of DAPI staining of WT (W303-1A and D273-10B/A1) and of their derivate deleted mutants (W gcn5Δ and D gcn5Δ) grown overnight in YP 2% glucose containing medium. Scale bars: 0.5 μm. (B) qRT-PCR analysis of mtDNA level of the two WT and their gcn5Δ derivative strains as above, grown in YP 2% glucose containing medium. The ratio between nuclear DNA mean value and mtDNA mean value (OXI1/ACT1) was used to overcome the variability among samples caused by total DNA quality. Data derive from at least three independent experiments and statistical significance by Student's t-test is indicated. **P<0.01 for deleted versus WT strain.
Fig. 4.
Fig. 4.
In gcn5Δ strains, the decrease of mtDNA copy number is accompanied by deletion of specific regions of the molecule. (A) qRT-PCR analysis of mtDNA copy number of the WT (W303-1A) and W gcn5Δ subclones reported in Table 2, grown in YP 2% glucose containing medium. The ratio between nuclear DNA mean value and mtDNA mean value (OXI1/ACT1) was used to overcome the variability among samples caused by total DNA quality. (B) Electrophoretic analysis on 1X TBE, 2.5% agarose gel of amplified sequences of mtDNA from WT (W303-1A) and from three W gcn5Δ clones is reported in Table 2. Amplified genes indicated in white. *, unspecific amplified sequence from COB and OXI3 genes.
Fig. 5.
Fig. 5.
GFP fluorescence colocalization of Gcn5. (A,B) Fluorescence microscopy of the GCN5-GFP with the endogenous GCN5 fused with the GFP gene grown in YP 2% glucose (A) or 3% glycerol (B) containing media to stationary phase. The fluorescent signals of the GCN5-GFP clone transformed with pmtRFP plasmid overexpressing the mitochondrial Red Fluorescent Protein show colocalization between Gcn5 and mitochondria.
Fig. 6.
Fig. 6.
Subcellular and submitochondrial fractions indicate that Gcn5 protein is localized in the mitoplasts. (A) Whole cell lysate (Super) and mitochondrial (Mito) fractions of W303-1A GCN5-9Myc or untagged control (No-myc) strains grown in YP 2% glucose or 3% glycerol containing media were analysed by 10% SDS PAGE. Gcn5-myc, Ada2 and Por1 antibodies were used as nuclear and mitochondrial marks, respectively. (B) Purified mitochondria (Mito) and supernatant (Super) from rho+ cells were treated (+) or not (−) with Digitonin or swelling buffer and sonication (Swelling) to obtain mitochondrial fractions. After isolation of outer membrane and inner membrane space (OMM/IMS), mitoplasts were fractionated in inner membrane (IMM) and matrix. Pellet (P) and soluble (S) fractions were isolated by ultra centrifugation and immunostained with antibodies against the Gcn5-myc, EF-Tu and Por1 for marks of different mitochondrial compartments (see Materials and Methods for details).
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