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First published online 13 May 2008
doi: 10.1242/jcs.028605

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Mitochondrial nucleoids undergo remodeling in response to metabolic cues

Martin Kucej^1,*, Blanka Kucejova^1,, Ramiah Subramanian², Xin Jie Chen^1, and Ronald A. Butow¹

¹ Department of Molecular Biology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA
² Department of Pathology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9148, USA

View larger version (44K): [in this window] [in a new window]   Fig. 1. Abf2 associates preferentially with complex GC-rich sequences. (A) Southern blot hybridizations of blots of mtDNA digested with DraI with the following probes: CsCl-purified total mtDNA sheared by sonication (left panel), DNA coimmunoprecipitated with an anti-Myc antibody from the strain expressing Abf2-13Myc fusion protein (middle panel), and DNA coimmunoprecipitated with an anti-Myc antibody from the control strain expressing untagged Abf2 (right panel). (B) The median-percentile rank analysis of ChIP DNA microarray hybridizations (supplementary material Fig. S3). All data points were sorted from highest to lowest Cy5:Cy3 ratios and each data point was assigned a percentile rank. The median-percentile ranks of each mtDNA region from three Abf2-13Myc and four Abf2 ChIP experiments were compared by subtracting the control medians (Abf2) from the sample medians (Abf2-13Myc). The gray peak represents the median-percentile rank difference of all mtDNA regions examined with the microarrays. The dark-gray peak shows median-percentile ranks of all the complex GC-rich sequences in the dataset; a bias to higher median-percentile ranks is evident. Therefore, these sequences were enriched in Abf2-13Myc ChIP sample. (C) A list of all DNA spots consistently enriched in the Abf2-13Myc ChIP-chip experiments. (D) Circular map of the intronless mtDNA. The inner black markers identify sequences covered by mtDNA microarray analysis. The innermost gray markers identify the regions significantly enriched in the Abf2-13Myc ChIP-chip experiments.

View larger version (59K): [in this window] [in a new window]   Fig. 2. Abf2 protein interactions in mitochondria. (A) Identification of mitochondrial Abf2-interacting proteins. Purified mitochondria from yeast strains expressing Abf2 and Abf2-13Myc were crosslinked with DSP. Mitochondrial lysates were incubated with DNaseI to remove mtDNA, and Abf2 protein was immunoprecipitated with anti-Myc antibody. Protein samples were compared on silver-stained gels. Protein bands that were unique to the Abf2-13Myc sample were further examined. (B) Silver-stained 4%-20% gradient SDS-PAGE gel of the protein samples immunoprecipitated with an anti-Myc antibody from the mitochondria isolated from MCC109 cells expressing the wild-type Abf2 protein and the Abf2-13Myc fusion protein. Where indicated, mitochondria were incubated with the protein crosslinker DSP prior to incubation with the antibody. a, Kgd1; b, Aco1; c, Ald4; d, Abf2-13Myc; e, Idh2; f, Idh1. (C) Southern blot confirming that the DNA was degraded after incubation with DNAseI in samples used for coimmunoprecipitation in B. DNA was isolated from 5 µg of purified mitochondria and from an aliquot of DNAseI-treated lysate that contained 50 µg of proteins. MtDNA was probed using a PCR-amplified region of the mitochondrial COX2 gene.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Mitochondrial nucleoids undergo remodeling. (A-C) Quantification of micrococcal-nuclease-sensitivity assays of (A) 21S_rRNA, (B) ATP6 and (C) intergenic region 81 using 1 mg of toluene-permeabilized mitochondria isolated from BY4741 cells grown in glucose-containing () and glycerol-containing () medium. Representative autoradiograms are shown in supplementary material Fig. S4. (D) Western blot of Pgk1, Abf2 and Cox2 proteins from cell extracts of BY4741 cells grown in YPD (glu) and YPG (gly) medium. Because expression of Pgk1 is not dependent on a carbon source (Roberts and Hudson, 2006), an anti-Pgk1 antibody was used to estimate the protein loading. Unlike Cox2 – the expression of which is much higher in glycerol than in glucose medium – Abf2 levels were comparable in both YPD and YPG. (E) Relative mtDNA copy number in BY4741 cells grown in YPD (glucose) and YPG (glycerol) medium assessed by Southern blot hybridizations using probes derived from the mitochondrial 21S_RNA and nuclear ACT1 genes. Error bars, average deviations.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Distribution of nucleoid proteins in sucrose gradients. Sucrose gradients of an equal amount of lysed purified mitochondria (1 mg of proteins) isolated from BY4741 cells grown in glucose and glycerol containing medium (A,B) and MCC109 cells grown in SD medium with isoleucine, leucine, valine or no amino acids (C,D) were collected in 16 fractions. Every fraction was probed for the presence of proteins Abf2, Mdh1, Hsp60, Aco1, Ilv5 (A,C) and mtDNA (B,D). Mdh1 is a matrix protein, which represents the control for the total lysis of mitochondria and the presence of matrix contamination. The combined occurrence of Abf2 and mtDNA indicates gradient fractions, which contain nucleoids. (A) Hsp60 is the only protein increased in fractions containing nucleoids, when cells are grown in glucose medium compared with glycerol medium. (C) Ilv5 is the only protein increased in fractions containing nucleoids, when cells are grown in medium without amino acids compared with medium containing isoleucine, leucine and valine.

View larger version (29K): [in this window] [in a new window]   Fig. 5. Distribution of Hsp60 A144V mutant protein in sucrose gradients. Sucrose gradients of an equal amount of lysed purified mitochondria (1 mg of proteins) from W303-1B cells expressing the wild-type Hsp60 protein or the Hsp60 A144V mutant grown in glucose and glycerol-containing medium were collected in 16 fractions. The gradient fractions were probed for the presence of proteins Hsp60, Abf2, Mdh1, (A) and mtDNA (B). Mdh1 is a matrix protein, which represents the control for the total lysis of mitochondria and the presence of matrix contamination. The combined occurrence of Abf2 and mtDNA indicates gradient fractions that contain nucleoids. An increased presence of Hsp60 A144V mutant protein in fractions containing nucleoids is visible. A much longer exposure time is required to detect the wild-type Hsp60 protein associated with nucleoids (compare with Fig. 4A).

View larger version (11K): [in this window] [in a new window]   Fig. 6. A model of mitochondrial nucleoid remodeling. In repressed conditions (growth in glucose medium), mitochondrial nucleoids form a more compact conformation with an increased ratio of Abf2 to mtDNA. Hsp60 is recruited to nucleoids. In conditions that activate respiration (growth in glycerol medium), an increased mtDNA copy number causes a decrease in the ratio of Abf2 to mtDNA. Nucleoids form a more open structure. In cells starved of amino acids, Ilv5 is recruited to nucleoids.

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