A novel DNA-binding protein bound to the mitochondrial inner membrane restores the null mutation of mitochondrial histone Abf2p in Saccharomyces cerevisiae

Abstract

The yeast mitochondrial HMG-box protein, Abf2p, is essential for maintenance of the mitochondrial genome. To better understand the role of Abf2p in the maintenance of the mitochondrial chromosome, we have isolated a multicopy suppressor (YHM2) of the temperature-sensitive defect associated with an abf2 null mutation. The function of Yhm2p was characterized at the molecular level. Yhm2p has 314 amino acid residues, and the deduced amino acid sequence is similar to that of a family of mitochondrial carrier proteins. Yhm2p is localized in the mitochondrial inner membrane and is also associated with mitochondrial DNA in vivo. Yhm2p exhibits general DNA-binding activity in vitro. Thus, Yhm2p appears to be novel in that it is a membrane-bound DNA-binding protein. A sequence that is similar to the HMG DNA-binding domain is important for the DNA-binding activity of Yhm2p, and a mutation in this region abolishes the ability of YHM2 to suppress the temperature-sensitive defect of respiration of the abf2 null mutant. Disruption of YHM2 causes a significant growth defect in the presence of nonfermentable carbon sources such as glycerol and ethanol, and the cells have defects in respiration as determined by 2,3,5,-triphenyltetrazolium chloride staining. Yhm2p may function as a member of the protein machinery for the mitochondrial inner membrane attachment site of mitochondrial DNA during replication and segregation of mitochondrial genomes.

FIG. 1

Functional deletion mapping of a 2.6 kb DNA fragment of clone D3. (A) Restriction map of the genomic insert of clone D3. The arrow indicates the YHM2 ORF. The two putative TATA boxes (−54 and −80) and the putative HAP2-HAP3-HAP4 transcription factor-binding site (−559) are indicated. (B) Deletion analysis of the D3 sequence. To identify the gene of interest, the fragment between the two NcoI sites of YHM2 was deleted. The plasmid with the deletion (pY-D3Δ935–1085) was tested for its ability to complement the temperature-sensitive phenotype of abf2 mutant cells. (C) Clone D3 can suppress the temperature-sensitive defect of the abf2 mutant but not the deleted clone D3Δ935–1085. The abf2 mutant cells transformed with YEp13, pY-D3, and pY-D3Δ935–1085 were grown to late log phase in synthetic medium (−Ura, −Leu) containing glucose and were streaked onto YPG plates (3% glycerol as nonfermentable carbon source) and grown at 30 or 37°C.

FIG. 2

The KLR17-3d (abf2::URA3) strain grown in the presence of glucose loses its mitochondrial genome, whereas the KLR71 (abf2::URA3, pY-D3) strain maintains [rho⁺] mitochondria. Cells were grown to stationary phase on YPD (2% glucose), streaked on YPDGE (1% yeast extract, 2% peptone, 0.1% glucose, 3% glycerol, 2% ethanol) plates, and then incubated at 30°C. The cells from large colonies of each strain were picked and stained with a DNA-specific dye, DAPI, and visualized by fluorescence microscopy (middle) and phase-contrast microscopy (left). Magnification, ×400. The major fluorescence source in the central region of the cells corresponds to DAPI-stained nuclear DNA. Small and weak fluorescent spots corresponding to mtDNA in the periphery are observed only in cells of KLR71 (abf2::URA3, pY-D3).

FIG. 3

Alignment and hydropathy plot of the deduced amino acid sequence of YHM2. (A) Multiple-sequence alignment for maximal amino acid similarities between Yhm2p and the members of the mitochondrial carrier family obtained with the ClustalW program in MacVector 6.01. The AAC2 gene encodes the mitochondrial ADP/ATP carrier (29), and ACR1 encodes a succinate/fumarate carrier (39). MRS3 encodes the suppressor of the mtRNA splicing defect (55, 56), and RIM2 encodes the suppressor of the pif1 mutant (50). YHM1 was isolated as a suppressor of the abf2 mutant (21, 34). The putative transmembrane (TM) domains are indicated, and the conserved motifs of mitochondrial energy transfer proteins are underlined. (B) Hydrophobicity profile of Yhm2p. The Yhm2p amino acid sequence was analyzed with the DNA Strider 1.2 program by the Kyte and Doolittle method. The most hydrophilic region (ILP3) of YHM2 is indicated.

FIG. 4

Yhm2p is a mitochondrial inner membrane protein. (A) Western blot analysis of the mitochondrial preparation obtained from wild-type yeast strain W303 shows the presence of Yhm2p in mitochondria. The proteins in whole cells, cytosol, and mitochondria were separated by SDS-PAGE and analyzed by immunoblotting with affinity-purified anti-Yhm2p antibody as described in Materials and Methods. (B) The purified mitochondria was extracted with 100 mM Na₂CO₃ (pH 12) or 1% Triton X-100, and the proteins in the supernatant and pellet were analyzed in panel A. The result shows that Yhm2p is an integral membrane protein. T, total mitochondria; S, supernatant; P, pellet. (C) Yhm2p is associated with the inner mitochondrial membrane, as determined by immunoblot analysis of inner mitochondrial membrane fractions (IM) and outer mitochondrial membrane fractions (OM) from the wild-type strain with antibodies against Yhm2p (α-Yhm2p), CoxIIIp (α-CoxIII), and porin (α-porin).

FIG. 5

DNA-binding ability of Yhm2p. (A) Construction of plasmids for expression of full-length and partial fragments of Yhm2p. A hexahistidine peptide sequence was placed at the amino terminus for affinity purification of the protein. For expression of a short segment of ILP3 (from 236 to 275 of Yhm2p [Fig. 3]), the ILP3 sequence was fused with GST. (B) Coomassie blue staining pattern of the proteins purified by affinity chromatography as described in Materials and Methods (left). DNA binding to the proteins immobilized on a nitrocellulose membrane (right). The purified recombinant proteins were fractionated by PAGE and transferred to nitrocellulose membrane. The immobilized protein was refolded in situ and probed with ³²P-labeled DNA as described in Materials and Methods. DNA bound to full-length Yhm2p and the carboxy-terminal half of the protein (Yhm2-ΔNp) but not to the amino-terminal half (Yhm2-ΔCp). (C) The amino-acid sequence of ILP3 of Yhm2p is similar to the amino-terminal sequences of various HMG boxes. The proline repeat in bold is important for the DNA-binding activity of the HMG box. (D) Coomassie blue staining of the purified recombinant GST-ILP3 protein. (E) DNA-binding activity of the ILP3 region. DNA binding to the ILP3 region was demonstrated by a DNA mobility shift assay as described in Materials and Methods.

FIG. 6

The putative DNA-binding domain is essential for suppression of the abf2 mutant. (A) Schematic representation of wild-type (YHM2) and mutant (Yhm2-PRM) Yhm2p. The proline repeat sequence in the putative DNA-binding subdomain was changed to pentaglycine by PCR-based site-directed mutagenesis (see Materials and Methods). (B) The putative DNA-binding subdomain is important for Yhm2p binding to DNA. The wild-type (WT) and mutant (PRM) Yhm2p were analyzed for their DNA-binding activity by Southwestern blotting as described in the legend to Fig. 5B. The same amount of each protein (1 μg) was used for both SDS-PAGE (12% polyacrylamide) and Southwestern analysis. To test the difference of DNA-binding affinity between Yhm2p and Yhm2-PRMp, the concentration of KCl in the binding buffer was varied from 50 to 150 mM. The mutant Yhm2-PRMp did not bind DNA under any conditions. (C) The mutation in the putative DNA-binding subdomain abolishes the suppression of the abf2 mutant. KLR17-3d (abf2::URA3) was transformed with the high-copy-number episomal plasmid harboring the yhm2-PRM gene (pY-D3PRM) and tested for growth on YPG at 30 or 37°C. KLR73 (abf2::URA3, pY-D3PRM) grew very poorly at both temperatures. The control strain, KLR71 (abf2::URA3, pY-D3), grew at both temperatures.

FIG. 7

Yhm2p is associated with mitochondrial nucleoids. (A) Sucrose gradient centrifugation of mitochondrial nucleoids. Mitochondria from wild-type yeast cells were lysed in 0.5% NP-40 and centrifuged. The pellet was loaded onto a sucrose gradient and centrifuged. The peak fraction containing mtDNA was reloaded onto a sucrose gradient and centrifuged as described in Materials and Methods. The gradient fractions were analyzed for mtDNA and protein by Southern blotting and the Bradford assay, respectively. (B) The protein profiles of the fractions from second sucrose gradient centrifugation were analyzed by SDS-PAGE (15% polyacrylamide). SM, size markers; the numbers indicate fraction numbers. The presence of Yhm2p and CoxIIIp in the purified mitochondrial nucleoids was determined by Western blot analysis. The anti-CoxIIIp antibody was used as a negative control. As shown in Fig. 4, the anti-CoxIIIp antibody used in this study was active and detected CoxIIIp in the inner mitochondrial membrane fraction.