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Corresponding author: Ronald A. Butow, Department of Molecular Biology and Oncology, University of Texas SW Med. Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9148, butow{at}swmed.edu (E-mail).
Communicating editor: K. J. NEWTON
| ABSTRACT |
|---|
Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf 2p, of Saccharomyces cerevisiae influences the stability of wild-type (
+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf 2p in mtDNA transactions. We find that in homozygous
abf 2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf 2p is known to be required for the maintenance of mtDNA in
+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in
- cells grown on the same medium. The content of both
+ and
- mtDNAs is increased in cells by 50150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf 2p plays a role in mtDNA copy control. Overproduction of Abf 2p by
10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of
+ mtDNA and a quantitative conversion of
+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf 2p in
- cells also leads to a loss of mtDNA, but at a slower rate than was observed for
+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf 2p because a mutant Abf 2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements
abf 2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf 2p and green fluorescent protein.These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination.
THE segregating unit of mitochondrial DNA (mtDNA) is generally believed to be a proteinDNA complex that can be visualized in cells with DNA-specific dyes as punctate-staining, cytoplasmic structures termed nucleoids or chondriolites (![]()
![]()
Mitochondria of wild-type yeast cells contain an abundant 20-kD protein that plays an important role in mtDNA maintenance. This protein was first identified by CARON et al. and called HM. The gene was later cloned and named ABF2 by ![]()
abf 2) is a loss of wild-type (
+) mtDNA from cells grown on rich dextrose medium. They observed, however, that when
abf 2 cells were grown on medium with glycerol, a nonfermentable carbon source,
+ mtDNA could be maintained indefinitely. These findings indicate that Abf 2p is not essential for mtDNA replication or gene expression.
Abf2p is a member of the family of high mobility group (HMG) proteins and it contains two HMG box domains. Because a truncated protein containing only one HMG box supplies the Abf 2p function for mtDNA stability, probably only one is essential (![]()
![]()
+ mtDNA in
abf 2 cells can be suppressed by other HMG proteins, including the yeast nuclear protein NHP6A (![]()
![]()
abf 2 cells (![]()
abf 2 cells can be partially suppressed by overexpression of the mitochondrial enzyme acetohydroxy acid reductoisomerase, the product of the ILV5 gene, which functions in branched chain amino acid biosynthesis (![]()
abf 2 phenotype by acetohydroxy acid reductoisomerase, which has no obvious DNA-binding motifs, is unknown.
Recent studies have suggested that
+ mtDNA in
abf 2 cells grown on glycerol medium is organized differently than in wild-type ABF2 cells (![]()
+ mtDNA in glycerol-grown
abf 2 cells stains more diffusely with the DNA-binding dye 4',6'-diamino-2-phenylindole (DAPI) than does
+ mtDNA in ABF2 cells, which shows a characteristic bright, punctate staining pattern. Second, some mtDNA sequences are four- to fivefold more sensitive to digestion by DNase I in permeabilized mitochondria from
abf 2 cells than in similarly prepared mitochondria from wild-type cells. Finally, a comparison of the protein profiles of purified mtDNA nucleoids isolated from
abf 2 and ABF2 cells showed that, in addition to the expected absence of Abf 2p, the nucleoids from
abf 2 cells lack polypeptides of 60 and 46 kD, both of which are present in amounts comparable to that of Abf 2p in nucleoids from wild-type cells. Together, these findings suggest that Abf 2p plays an important role in the organization of mtDNA.
To better understand the role of Abf 2p in mtDNA maintenance, we have analyzed the sorting, copy number, and transmission properties of
+ and
- mtDNAs in wild-type cells and in cells that either lack Abf 2p or contain elevated levels of the protein. Our experiments indicate that both
+ and
- mtDNA copy number can be modulated by the level of Abf 2p; however, very high levels of Abf 2p result in mtDNA instability, an effect that probably requires the DNA-binding properties of the protein. Surprisingly, unlike
+ mtDNA,
- mtDNAs are stable in
abf 2 cells when grown on rich dextrose medium. These results, together with data showing that mtDNA recombination is reduced in
abf 2 cells, suggest that Abf 2p influences both the replication and segregation of mtDNA, perhaps through a role in forming or stabilizing mtDNA recombination intermediates.
| MATERIALS AND METHODS |
|---|
Strains, growth media, and growth conditions:
Strains of S. cerevisiae used in this study are listed in Table 1. MAT
derivatives of 14WW and 14WW
abf 2 were made by mating-type switching induced by expression of the HO gene under galactose control (![]()
![]()
+
+ Cr Or and
+
+ Cs Os, with markers conferring resistance or sensitivity to chloramphenicol (C) or oligomycin (O) [see ![]()
o derivatives of strains were obtained by growth of cells in rich medium containing 2% dextrose and 25 µg/ml ethidium bromide. The mitochondrial genome of the HS40
- strain is a 760-bp repeat containing ori5, a putative origin of mtDNA replication (![]()
+ tester strains, >98% of the diploid progeny are
- petites with the HS40 mitochondrial genome. The mitochondrial genome of the
- VAR1 strain is a 2-kb repeat encompassing the VAR1 gene, which was first analyzed in strain 5D2-33 (![]()
- diploid progeny when crossed to
+ testers. These
- genomes were introduced into
14WW and
14WW
abf 2 by cytoduction; the desired cytoductants were identified by colony hybridization or by Southern blotting with petite genomespecific probes. All standard yeast genetic analyses were performed as described in ![]()
|
YP medium contains 1% yeast extract and 2% Bacto peptone, and either 2% dextrose (YPD), 2% glycerol (YPG), or 2% galactose (YPGal). YNB medium contains 0.67% yeast nitrogen base without amino acids and either 4% dextrose (YNBD), 4% dextrose and 1% casamino acids (YNBD+cas), 2% glycerol (YNBG), 2% glycerol and 1% casamino acids (YNBG+cas), or 2% galactose and 1% casamino acids (YNBGal+cas). All YNB media were supplemented with the requisite nutritional requirements. RG medium contains 0.2% yeast extract, 0.2% Bacto peptone, 0.05% NaCl, 0.1% (NH4)2 SO4, 0.05% MgCl2, 0.05% KH2PO4, pH 6.5, and 3.2% glycerol. Solid media contained 2% Bacto agar. Cells were grown at 30°. Colonies were scored as
+ or
- by the tetrazolium overlay method (![]()
+ and
- by the red colony color that
+ ade1 and ade2 strains develop.
Plasmids:
Plasmid DNAs were prepared by alkaline lysis using E. coli strains DH5
or XL1-blue.
pGCS1 is a CEN-URA3 plasmid containing the CIT1 coding region under control of the GAL1 promotor (AZPIROZ and BUTROW 1993). pAM1A20 is a pUC119-based ARS1-CEN4-URA3 plasmid containing the 1.6-kb EcoRI fragment of yeast DNA that includes the ABF2 gene (![]()
![]()
To construct plasmid pGAL68/ABF2, a 0.9-kb fragment of DNA containing the entire ABF2 coding sequence flanked by restriction sites 5'-BamHI and 3'-HindIII was generated by PCR using the primers 5'-GTAAACAGATTAACAAAGGATC CAATCAATTACAACAAC-3' and 5'-TCGTAAAGAAGCTTTG TAAAGGTGAGGACG-3'. The PCR product was digested with BamHI and HindIII, and the fragment containing the coding region of ABF2 plus 24 bp of 5' and 376 bp of 3' sequences was gel purified and ligated into the BamHI-HindIII site of pGAL68, which was previously called pSEYC68-Gal (![]()
Plasmid YIp356/ABF2 was constructed by cloning the 1.6-kb EcoRI fragment of plasmid pAM1A20 containing the ABF2 gene into the EcoRI site of plasmid YIp356 (![]()
![]()
Plasmid pRS416/Abf 2-GFP (CEN URA3) was constructed by ligating a 0.8-kb EcoRI-XhoI fragment containing the 5' untranslated region (UTR) and coding region of ABF2 in frame to a 0.73-kb XhoI-KpnI fragment containing the coding region of green fluorescent protein (GFP), followed by a 1-kb KpnI-EcoRI fragment of the ABF2 3' UTR, and cloned into the EcoRI site of pRS416. The mutant allele, abf 2-1-2- (see below), was cloned into pRS416 in the same way to yield plasmid pRS416/Abf 2-1-2--GFP. pRS416/CS1-GFP was constructed by ligating a 0.9-kb EcoRI-XhoI fragment of the CIT2 gene containing the 5' UTR and the region of the open reading frame encoding the first 52 amino acids of citrate synthase 1 (CS1) fused to the XhoI-KpnI fragment of the GFP-coding region, followed by a 0.5-kb KpnI-HindII fragment of the CIT2 3' UTR, and cloned into the EcoRI-HindIII site of pRS416.
Construction of mutant abf 2-1-2-:
The 1.6-kb EcoRI fragment from pAM1A20 containing the ABF2 gene was cloned into pRS416 (to generate pRSABF2) and site-specific mutations were placed in the two HMG boxes. Primers corresponding to box 1 (5'-TAAGAAATAAGCAGATGTGGGGGCGCC AGGAACCCTGTTTTATCAATTC-3') and box 2 (5' CTTATT GAAGGGTCCTGCTGGGCCGGCTGGAGGAAGTTTTTCGT CAAAC-3') were used to mutate Lys44Arg45 and Lys117Lys118 to GlyAla and AlaGly, respectively. Mutagenized plasmids (referred to as pRSabf 2-1-2-) were propagated in E. coli strain CJ236, and the presence of the mutations was monitored by screening for the NarI and NaeI restriction sites, respectively (underlined).
Yeast transformations were carried out by the lithium acetate method (![]()
Strain constructions:
YIp356/ABF2 was linearized at the NcoI site in the URA3 gene and transformed into strain 14WW
+; integration of the plasmid into the URA3 locus yielded strain 14WW/ABF2 with two genomic copies of the ABF2 gene. Plasmid YIp356/ABF2/TRP1 was linearized at an internal XbaI site in the TRP1 gene and integrated into the genomic TRP1 gene by transformation of strain 14WW/ABF2; the resulting strain, 14WW/2ABF2, has three genomic copies of the ABF2 gene. To generate strain 14WW/GAT, in which one copy of the ABF2 gene is under control of the GAL1 promoter, a 1.6-kb EcoRI-HindIII fragment of plasmid pGAL68/ABF2 containing the ABF2 gene under control of the GAL1 promotor was cloned into the multicloning site of plasmid YIp356, yielding YIp356/GA. A Klenow-filled BamHI fragment of plasmid YDP-W containing the TRP1 gene was cloned into a Klenow-filled HindIII site of plasmid YIp356/GA. The resulting plasmid, YIp356/GAT, was linearized at the NcoI site, gel purified, and transformed into strain 14WW
+. Ura+ and Trp+ transformants were selected, and integration was verified by Southern blot analysis.
Cells were cured of URA3 plasmids using standard methods (![]()
mtDNA recombination:
abf 2 strains 70 (MATa Cr Or) and 43 (MAT
Cs Os) and YCpABF2 transformants of those strains were grown on YPG medium and mated in the four possible combinations for 1216 hr on solid YPG medium. Diploids were selected by replica plating on appropriately supplemented YNBG medium. Diploid cells were then plated for single colonies on RG medium. Resistance or sensitivity to chloramphenicol (C) and oligomycin (O) was tested by replica plating to RG medium containing either 3 mg/ml chloramphenicol or 3 µg/ml oligomycin, and plates were scored after 4 days incubation at 30°.
Analysis of mtDNA and Abf2p content:
Total cellular DNA from the various yeast strains was prepared by the rapid glass bead method of ![]()
+ mtDNA, DraI for HS40
- mtDNA, or HincII for VAR1
- mtDNA, and was fractionated on 1% agarose gels. Blotted samples were hybridized with the following 5' 32P-end labeled probes: VAR1, 5'-ATGGTATCTTAAC TAATTATCAACGTA-3'; HS40, 5'-GATAAACAAGAAGATA TCCGGGTC-3'. For some experiments, HS40 DNA samples were hybridized with random-primed HS40 DNA (Random Primed DNA Labeling Kit; Boehringer Mannheim Biochemicals, Indianapolis) purified from strain 14WW
- HS40 and gel purified after cleavage with EcoRV. A 679-bp PCR-amplified fragment of the COXII gene (nt 13352014 of GenBank accession number J01485) was labeled by random priming and used as the probe for
+ genomes. For each digest, the level of the single-copy ACT1 gene was determined by hybridization with a random-primed fragment of the ACT1 gene [the 0.6-kb XbaI-PstI internal fragment from the ACT1 gene from pGEM-actin (![]()
The amount of Abf2p in whole-cell extracts was determined by FluorImager (Molecular Dynamics, Sunnyvale, CA) scanning of Western blots using a polyclonal antibody to Abf2p that was expressed and purified from E. coli as described (![]()
Staining and microscopy:
To analyze the sorting of the mitochondrial matrix protein CS1 and mtDNA in zygotes, fixed cells (4% formaldehyde for 1 hr at 30°) were washed in P buffer (40 mM KH2PO4, pH 6.5, and 0.5 mM MgCl2). Spheroplasts were prepared and placed on poly-L-lysinecoated multiwell slides as described in ![]()
![]()
o cells in cultures was determined by direct count of DAPI-stained cells. For microscopic analysis of GFP fusion proteins,
+ cells of strain 14WW were transformed with pRS416/ABF2-GFP, pRS416/abf 2-1-2--GFP, or pRS416/CS1-GFP, and were grown in YNBR+cas medium to midlogarithmic phase. Cells were collected by centrifugation and resuspended in sterile distilled water to a density of 5 x 106 cells/ml. A small drop of the cell suspension (23 µl) was placed on a microscope slide, covered with a cover slip (22 x 22 mm), sealed with 0.5% agarose, and observed under the microscope. Cells were observed by fluorescence with a cooled CCD (model C5810; Hamamatsu Photonic Systems, Bridgewater, NJ) camera on a DMRXE microscope (Leica, Deerfield, IL) equipped with the following filter set: excitation, 450490 nm; dichroic, 510 nm; barrier, 515 LP, an HBO 100W/2 mercury arc lamp, and a x100 Plan-Apochromat objective. Fluorescence and differential interference contrast (DIC) digitized images were acquired using Adobe Photoshop (Adobe Systems, Mountain View, CA).
| RESULTS |
|---|
Altered sorting of mitochondrial constituents in zygotes formed from
abf 2 parents:
We previously developed a system to follow the kinetics of sorting of mtDNA and mitochondrial matrix proteins in zygotes from synchronously mated cells (![]()
+ cells or between a
o petite and a
+ strain, and the sorting of the protein was then followed at different time intervals after synchronous zygote formation by indirect immunoflourescence using anti-CS1 antiserum. Sorting of mtDNA was determined either by pedigree analysis of the mitochondrial genotype of zygote end buds or by direct vizualization of mtDNA by DAPI staining.
An important finding of those experiments was that in a
+ x
+ cross, the matrix protein marker equilibrated throughout the zygotes after ~4 hr of zygote maturation while the
+ parental mtDNAs remained largely unmixed (detected as progeny of end buds that were homogeneous for one parental mtDNA or the other). In the
+ x
o cross, an intermediate in the sorting process was observed as a population of zygote forms in which the matrix marker protein, initially present only in the mitochondria of the
o parent, had quantitatively moved to the
+ end of the zygote before equilibrating with mtDNA throughout the zygote and into the emerging diploid bud. This unusual zygote form was called "asymmetic" (A). We concluded in those studies that A-form zygotes were not the result of the transfer of bulk mitochondria from the
o to the
+ end because the CS1 fluorescence and DAPI staining of mtDNA were colocalized in one end of the zygote. Although the mechanism accounting for this quantitative transfer of the matrix protein from the
o to the
+ end of the zygote is not known, the formation of A-form zygotes requires the presence of
+ mtDNA in one parent of the cross, although that parent need not be respiratory competent (![]()
The appearance of A-form zygotes in
o x
+ crosses, as well as the subsequent equilibration of matrix protein and mtDNA throughout the cell, which yields mixed, M-form zygotes, provides a convenient assay to determine whether Abf 2p plays a role in the sorting process. To examine this possibility, we first verified that the wild-type (ABF2) strains used in the present study show the same zygote-sorting properties described previously by ![]()
+ (CS1-) and 15WW
o (CS1+), each of which has a wild-type ABF2 allele. Unmixed, U-form zygotes containing CS1 in the
o end and mtDNA in the
+ end are present initially. By 4 hr after mating, the U-form zygotes have been largely replaced by mixed, M-form zygotes in which CS1 and mtDNA have equilibrated in the cell. Two intermediates in the mixing process are observed: first, partially mixed, P-form zygotes are present in which CS1 has begun to move to the
+ end; later, asymmetric, A-form zygotes are seen in which essentially all of the CS1 from the
o end has moved into the
+ end of the zygote and colocalizes with mtDNA. These zygote forms and the kinetic pattern of their appearance and disappearance are identical to those described previously using other strains (![]()
|
To determine whether the absence of Abf2p affects the sorting of mtDNA and matrix proteins, we repeated the
+ x
o cross with
abf 2 derivatives of the parental strains used above. As summarized in Figure 1B, no A-form zygotes were detected in the population of this homozygous
abf 2 cross. Instead, a small population of the P-D-form zygotes was observed in which CS1 from the
o end of the zygote and mtDNA from the
+ end moved toward each other and appeared colocalized in the neck region of the zygote. We have previously observed P-D-form zygotes only in crosses between
o and
- cells (![]()
o and
+ cells, even if the
+ cells were respiratory deficient. We previously interpreted the A-form zygotes as indicating the association of
+ mtDNA with a segregation apparatus, and the P-D-form zygotes as reflecting a poorer association of
- mtDNA with the putative segregation apparatus. Thus, the absence of A-form zygotes and the presence of P-D-form zygotes in crosses between
o and
+ cells lacking Abf 2p show that Abf 2p influences the distribution and mixing of mitochondrial DNA and protein in crosses. Finally, other than the differences in the sorting patterns noted above, we have not detected any obvious changes in the mitochondrial structure of
abf 2 cells.
mtDNA recombination is reduced in crosses between
abf 2 strains:
Recombination of mtDNA requires the mixing and subsequent pairing of parental mtDNA molecules in zygotes. The alterations in the "normal" pattern of both mtDNA and protein sorting in zygotes lacking Abf 2p described in the preceding section raise the possibility that mtDNA recombination might also be affected. To examine this, crosses were carried out with the
abf 2 strains 70 and 43 that either lacked or contained the CEN plasmid YCpABF2 encoding the wild-type ABF2 gene. The mtDNAs in these strains contain markers conferring either resistance (strain 70) or sensitivity (strain 43) to chloramphenicol and oligomycin (Cr Or and Cs Os, respectively). In standard crosses, these two markers yield the maximum level of ~25% recombination and, therefore, appear to be unlinked. The actual level of recombination observed, however, is lower than that maximum in so-called "biased" crosses where the parents have unequal inputs of mtDNA (![]()
Table 2 shows that the homozygous ABF2 cross (cross I) yields ~11% C-O recombinants. Similar values were obtained in the reciprocal heterozygous crosses (crosses II and III) where one parent is
abf 2 and the other is ABF2. In the homozygous
abf 2 cross (cross IV), however, recombination was reduced five- to sevenfold. Although there are differences in the input bias associated with the
abf 2 allele (see below), the comparison of crosses III and IV, where the parental allele outputs are essentially identical, shows clearly that the absence of Abf 2p suppresses mtDNA recombination. We conclude that Abf 2p is important for efficient recombination of mtDNA. These results and those of the zygote-sorting experiments above establish new phenotypes for the
abf 2-null allele.
|
- mtDNA is stable in
abf 2 cells:
The mitochondrial genomes of
- petite mutants have sustained large deletions of
+ mtDNA. Despite differences in the extent of deletion of
+ mtDNA sequences, the proportion of total cellular DNA represented by
- mtDNAs is comparable to that of
+ mtDNA (![]()
![]()
- mtDNAs are often stable in genetic backgrounds where
+ mtDNA is unstable. For instance, ![]()
- genomes are stably maintained in cells containing a null allele of RPO41, the gene encoding mitochondrial RNA polymerase, whereas
+ mtDNA is unstable in such cells. Those experiments underscore the well-documented but poorly understood observation that mitochondrial gene expression, including protein synthesis, is required for the maintenance of
+ mtDNA (![]()
- mtDNA, as it is for
+ mtDNA. Petites containing two different
- mitochondrial genomes (HS40 and VAR1) were analyzed. These
- genomes were chosen because they retain similar-sized small fractions of the
+ genome (0.82 kb) and have a similar A + T content; however, they have no sequences in common and they differ in the extent of suppressiveness (see MATERIALS AND METHODS for more details). Each
- mtDNA was transferred by cytoduction into cells of strain 14WW
abf 2, which contained the ABF2 gene on the plasmid YCpABF2. The plasmid was then cured from each strain, and the stability of the
- genomes was analyzed during growth of the strains on Y PD medium. As controls, these
- genomes were also introduced into the parent (ABF2) strain and tested for stability in the same way.
The ABF2 and
abf 2
- strains, grown in liquid Y PD, and the control
+ strains, grown in liquid Y PG, were inoculated into liquid Y PD and sampled at various times. Total cellular DNA was isolated from the samples, cleaved with the appropriate restriction enzymes, blotted, and hybridized with probes specific for the
+ and
- mtDNAs, as described in MATERIALS AND METHODS and in the legend to Figure 2. A specific probe to the single-copy nuclear ACT1 gene was included to allow normalization of the mtDNA content to that of nuclear DNA. Figure 2 shows a progressive decrease in
+ mtDNA level in the
abf 2 cells during growth on Y PD medium. The time course of loss of
+ mtDNA has not been analyzed previously, and it is worth noting that the
+ mtDNA is lost at a rate that is slower than would be expected if cells, upon shift to Y PD medium, immediately failed to replicate their mtDNA. If that were the case, by six generations of growth on Y PD medium, the population would have contained only 12% of the normalized mtDNA content of the starting population, which was clearly not observed in the data of Figure 2. In contrast to these results for
+
abf 2 cells, neither the HS40 nor the VAR1
abf 2
- strains had any appreciable decrease in mtDNA content relative to the ACT1 signal when grown on Y PD medium for the same number of generations as the
+ strain. Indeed, we find that these
- mtDNAs can be maintained indefinitely in
abf 2 strains grown on Y PD medium. Inspection of the data of Figure 2 also shows that there is no significant difference between the ABF2 and
abf 2 strains in the amount of the HS40 and VAR1
- mtDNAs. On the other hand, as seen in the lanes marked "0 Generations," glycerol-grown
+
abf 2 cells have about half as much mtDNA as do
+ ABF2 cells (see below). Together, these data show that
- mtDNAs respond differently to a lack of Abf 2p than does
+ mtDNA both in terms of mtDNA stability and copy number.
|
The ABF2 gene dosage influences mtDNA copy number:
To investigate further the influence of Abf 2p on
+ mtDNA copy number, we examined the effects of increased dosages of ABF2 on the amount of
+ mtDNAs in cells grown on glycerol or dextrose medium, and on the levels of VAR1 and HS40
- mtDNAs in cells grown on dextrose medium. For these experiments, we analyzed derivatives of
+ strain 14WW (ABF2) carrying an average of two extra copies of the ABF2 gene on a low copy number plasmid, YCpABF2 (based on plasmid copy number estimates, data not shown), or two extra copies of ABF2 integrated into the nuclear genome, one near the TRP1 gene and another near the URA3 gene. We analyzed derivatives of the
- strains containing YCpABF2. Aliquots from logarithmic phase cultures of these strains were removed, and the amount of mtDNA was determined by quantitative Southern blot analysis. Figure 3A and Figure B, are representative experiments showing that increasing the gene dosage of ABF2 by two- to threefold (and Abf 2p in parallel, as determined by dot blot analysis) results in a 50150% increase in the amount of mtDNA. Similar results were obtained when these cells were grown in dextrose medium. In six independent experiments that we have carried out with the
+ and
- strains with the integrated or plasmid-borne extra copies of ABF2, the overall average increase in mtDNA copy number was 100%, ranging from 30 to 330%, with no discernible difference between the
+ and
- mtDNAs. The data of Figure 3 also show that the
+ mtDNA content is reduced ~70% in these
abf 2 cells grown in Y PG medium, as was evident in an independent experiment shown in Figure 2. This decrease is not likely to be caused by a large accumulation of
o petites in the population because >95% of colonies of
abf 2 cells pregrown on glycerol medium contain some
+ cells when grown on medium containing dextrose (data not shown). Thus, we conclude that ABF2 not only plays a role in the maintenance of
+ mtDNA, but also influences the amount of mtDNA in both
+ and
- cells.
|
High levels of ABF2 expression result in loss of
+ and
- mtDNA:
![]()
+ cells of strain 14WW containing an extra copy of ABF2 under the control of the GAL1 promoter integrated near the URA3 gene. This strain, 14WW/GAT, and the parental strain, 14WW, were grown in Y PG medium, shifted to Y PGal, and monitored for the formation of petites by direct plating on Y PD medium, for the level of mtDNA by quantitative Southern blotting, and for the amount of Abf 2p by Western blot analysis.
Figure 4 shows that after the shift of the 14WW/GAT cells from Y PG to Y PGal medium, the Abf 2p content increases by 810-fold by two generations and by about another twofold after a total of four generations. Coincident with the increase in Abf 2p is a rapid decrease of mtDNA content and a concomitant production of petite mutants, which account for 100% of the cell population by four generations. Inspection of the kinetics of decrease in mtDNA content suggests that net synthesis of mtDNA ceases at high levels of Abf 2p because the normalized loss of mtDNA roughly follows cell doublings. No mtDNA is detected by DAPI staining of the petite colonies (data not shown), indicating that this regime induces
o petites. Growth of the parental strain, 14WW (having just one copy of the ABF2 gene), in Y PGal medium caused no increase in Abf 2p, no induction of petite mutants, and no loss of mtDNA other than what can be accounted for by the shift from glycerol to galactose medium. These experiments show that although moderate increases in Abf 2p result in an increase in the copy number of
+ mtDNA (Figure 3), larger increases have the opposite effect, causing a striking failure of mtDNA maintenance that leads to petite formation.
|
To determine whether hyperexpression of ABF2 also destabilizes
- mtDNA, we introduced the HS40
- mitochondrial genome by cytoduction to yield strain 14WW/GAT(
- HS40) with an extra copy of ABF2 under galactose control. These cells were grown in Y PD medium, transferred to Y PGal medium, and monitored for the level of HS40 mtDNA by Southern blot hybridization. Figure 5 shows that after a brief lag, there is a progressive decrease in the ratio of
- HS40 mtDNA to nuclear DNA during the time course of Abf 2p overproduction, with the mtDNA content falling to ~20% of the starting level after six generations of growth on galactose. A comparison of the data of Figure 4 and Figure 5 shows that the rate and extent of HS40 mtDNA loss is lower than for
+ mtDNA: the amount of mtDNA in
+ cells has dropped by >50% between one and two generations of growth on galactose, whereas there is very little decrease in
- mtDNA during the first two generations of growth of the petite strain on galactose. These data suggest that the reduction of mtDNA relative to nuclear DNA is caused by an inhibition of net mtDNA synthesis by the excess Abf 2p.
|
Loss of mtDNA caused by high levels of Abf 2p is relieved by mutations of the DNA-binding motifs of the HMG boxes of Abf 2p:
One can envisage that the loss of mtDNA in cells overproducing Abf 2p is caused by some indirect effect on mitochondrial biogenesis. For example, the large increase in Abf 2p synthesis may saturate some component of the mitochondrial protein import system, which then could conceivably lead to an imbalance of intramitochondrial components that are necessary for replication and maintenance of mtDNA. Alternatively, a large increase in the amount of Abf 2p could have a direct effect on the ability of mtDNA to be propagated. In that case, the deleterious effect of a large dose of Abf 2p would be expected to depend directly on its DNA-binding properties. To distinguish between these possibilities, we made site-directed mutations in key residues of both HMG box domains of Abf 2p that are expected to compromise the ability of the protein to bind to DNA, and we tested effects of that mutant allele on the stability of mtDNA.
A highly conserved motif near the N terminus of HMG boxes contains a Pro followed by one or two basic residues (![]()
![]()
![]()
abf 2
+ cells in the C EN plasmid pRSabf 2-1-2-, and its expression was compared with that of the wild-type ABF2 allele, which is also expressed in the same strain from the same C EN vector. Western blot analysis of extracts from these cells grown on Y PG medium shows that the wild-type and mutant proteins accumulate to comparable levels (Figure 6B).
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We next determined whether Abf 2p-1-2- complements the mtDNA instability phenotype of
abf 2 cells. Derivatives of strain 14WW
abf 2
+ with ABF2 or abf 2-1-2 - on a CEN plasmid, as well as a control strain transformed with vector pRS416 and lacking the insert, were constructed. Cells were grown on Y NBG+cas medium and then transferred to liquid Y NBD+cas medium, grown for ~15 generations, and aliquots were plated directly on Y PD medium. After 3 days growth, the colonies were scored as
+ or petite using the tetrazolium overlay agar method, in which colonies of respiratory-competent
+ cells stain red, while those of respiratory-deficient petite cells remain white (![]()
abf 2 cells were petite (white; plate 1), whereas only ~5% of the colonies derived from
abf 2 cells containing the wild-type ABF2 allele were petite (plate 2). By contrast, ~80% of the colonies derived from cells containing the abf2-1-2 - allele were petite (plate 3), comparable to the
abf 2 cells transformed with the vector pRS416 alone (plate 4). These results show that Abf 2p-1-2-, although present at a comparable level as Abf 2p, is substantially defective because it only weakly complements the mtDNA instability phenotype of
abf 2 cells.
Although these HMG box mutations in Abf 2p-1-2- were chosen on the basis of the reduced in vitro DNA-binding capacity of the rat HMG1 polypeptide (![]()
To address this question, we constructed expression plasmids encoding fusion proteins between the C-terminal end of full-length Abf 2p, the mutant protein Abf 2p-1-2-, and GFP so that the tagged proteins are expressed from the natural promoter of the ABF2 gene and targeted to mitochondria. As a control, another construct was made between the promoter and sequences encoding the first 52 amino acids of CS1 (which contains the matrix-targeting presequence) and coding sequences of GFP. Cells of strain 14WW
+ were transformed with a plasmid carrying each gene fusion, and the intracellular localization of the resulting fusion proteins was examined by epifluorescene microscopy. Figure 7 (top panel) shows a typical result for Abf 2p-GFP: this fusion protein shows a bright, punctate staining pattern that colocalizes with the punctate, DAPI staining of mtDNA. In contrast, Abf 2p-1-2--GFP (middle panel) is localized diffusely throughout the mitochondrial reticulum in a pattern that is more similar to that of CS1-GFP (bottom panel). These results strongly suggest that while most, if not all, of the Abf 2p-GFP is associated with mtDNA in vivo, the vast majority of Abf 2p-1-2--GFP is not bound to mtDNA and is probably localized in the mitochondrial matrix.
|
We next tested whether overexpression of abf 2-1-2- causes the loss of
+ mtDNA. To insure comparable retention of plasmids containing the wild-type ABF2 and mutant abf 2-1-2- alleles under control of the GAL1 promoter, galactose induction was carried out in selective Y NBGal+cas medium. The experiment of Figure 8 shows that, after a shift of cells from glycerol to galactose medium, both the wild-type and mutant proteins are induced with similar kinetics and reach the same maximum level after about three generations. As seen in Figure 8, cells overexpressing wild-type Abf 2p rapidly produce petite mutants, such that by two generations only ~5% of the population is
+. In contrast, at two generations, >70% of the cells in the culture overexpressing the mutant protein are still
+, and even by six generations, ~60% of the cells are
+. Thus, despite comparable kinetics and levels of induction of the wild-type and mutant proteins, these mutations in the Abf 2p HMG boxes that are involved in DNA binding largely suppress the production of petites when the mutant protein is overexpressed.
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| DISCUSSION |
|---|
Abf 2p has been suggested to play a structural role in mtDNA metabolism through its ability to bend and wrap DNA (![]()
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+ and
- mtDNAs for Abf 2p.
The absence of Abf 2p affects mtDNA recombination and the sorting of mitochondrial constituents in crosses:
We have previously characterized the pattern of sorting of mitochondrial matrix protein and mtDNA in zygotes derived from parental strains with different mitochondrial genotypes, particularly the mixing of these mitochondrial constituents in
0 by
+ crosses (![]()
0 by
+ cross such that it resembles the sorting pattern previously described for
0 by
- crosses, namely the loss of A-form zygotes and the appearance of the PD form, in which matrix protein and mtDNA are concentrated in the neck region of the zygote. Those results suggested that the
- mtDNA movement through the zygote was altered with respect to
+ mtDNA. In addition, although we do not yet understand the mechanism accounting for the vectorial movement of matrix protein in
o by
+ crosses that gives rise to the A-form zygotes, the fact that this form is absent in
abf 2 zygotes and that the PD form is detected suggests that the absence of Abf 2p may have a similar effect in altering the movement of
+ mtDNA.
We have also shown that the absence of Abf 2p markedly reduces the efficiency of mtDNA recombination. Because the extent of mtDNA recombination must clearly depend on the extent to which parental mtDNA molecules mix in the zygote, it is conceivable that the reduced level of recombination observed in the cross between
+
abf 2 parental strains could be caused by some effect on mtDNA mixing. Earlier genetic data showed that the mixing and recombination of mtDNA is restricted largely to the medial portion of the zygote, where the parental mitochondria presumably fuse (![]()
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Variations in the content of Abf 2p affect the amount and stability of mtDNA:
In
abf 2
+ cells maintained on glycerol medium, the mtDNA content is reduced by roughly 50% compared with ABF2 cells, whereas in
+ and
- cells carrying several extra copies of the ABF2 gene, the amount of mtDNA increases typically by 50150%. These findings imply that the amount of mtDNA could be regulated through regulation of Abf 2p levels. Little is known about the regulation of Abf 2p levels in mitochondria, although it has been shown that Abf 2p mRNA abundance is not repressed by glucose in cells in which there is repression of the
+ mtDNA content (![]()
10-fold the wild-type amount) result in a failure of both
+ and
- mtDNAs to be propagated. These findings confirm and extend the observation reported by ![]()
+ cells from a galactose-inducible promoter.
Our results strongly suggest that the dramatic instability of mtDNA in cells with high levels of Abf 2p depends on the interaction of the protein with mtDNA. We constructed and characterized a new allele of the ABF2 gene with mutations in a highly conserved motif of both HMG boxes. Analogous mutations in a polypeptide fragment of a mammalian HMG1 protein reduced its binding to cruciform DNA by ~10-fold (![]()
abf 2 cells. And when overexpressed in
+ cells, Abf2-1-2-p is much less effective than Abf2p in inducing petites.
Functions of Abf 2p in the maintenance of mtDNA:
One possibility to account for some of the observations described here is that Abf 2p functions directly in mtDNA replication as a transcription factor for the generation of RNA primers. This would be analogous to the role of the human mitochondrial homologue of Abf 2p, h-mtTFA, in promoter activation of human mtDNA (![]()
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It has also been shown that
- mtDNAs can be maintained in cells lacking the product of the nuclear RPO41 gene (![]()
+ mtDNA (![]()
+ mtDNA gene expression at many different levels results in instability of the mitochondrial genome (![]()
![]()
+ mtDNA. It is conceivable that
- mtDNAs replicate by a mechanism entirely different from that of
+ mtDNA, but definitive evidence supporting that view is lacking. Finally, recent studies showing that hypersuppressivitythe preferential transmission in crosses of
- mtDNAs containing putative origin sequencesis maintained in the absence of the RPO41 gene product (![]()
An alternative possibility is that Abf 2p affects mtDNA stability and copy number through a function in recombination. This view is consistent with the following observations: (1) HMG box proteins, including Abf 2p, bind preferentially to cruciform DNA (![]()
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The yeast mitochondrial genome is very active in recombination, where 1% recombination corresponds to a mtDNA interval of ~100 bp. A significant fraction of yeast mtDNA in wild-type cells is known to exist as larger than unit size molecules (![]()
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An important relationship between recombination intermediates and mtDNA transmission has been established from the studies of ![]()
![]()
- mtDNA. The mechanism of hypersuppressivity is not known, but it is generally thought to result from outcompetition by hypersuppressive
- mtDNA of
+ mtDNA in zygotes of hypersuppressive
- x
+ crosses. Although the effect of loss of the MGT1 product on the stability of
+ mtDNA was more subtle than the dramatic loss of hypersuppressivity in hypersuppressive
- x
+ crosses, the overall conclusion of those studies was that the requirement to cleave recombination intermediates in an MGT1-dependent reaction affects transmission of mtDNA during mitotic growth and in crosses.
The observed decrease in
+ mtDNA copy number in
abf 2 cells grown on glycerol medium may result from a reduction in the steady-state level of recombination intermediates, whereas in cells containing greater than wild-type levels of Abf 2p, more mtDNA would be present as recombination structures. By stabilizing recombination intermediates, increased Abf 2p levels would effectively increase the recombination-dependent priming of mtDNA replication that leads to higher levels of mtDNA. In the extreme case of cells with
10-fold higher levels of Abf 2p, however, most of the mtDNA might be trapped as networks of recombination structures. That such structures could lead to a rapid production of
o petites is suggested by the studies of ![]()
+ mtDNA (![]()
It is possible that the dramatic effect of high levels of Abf 2p on mtDNA replication and stability may result from the general DNA-binding activity of Abf 2p rather than or in combination with