Isolation of the mitochondrial nucleoids from yeast Kluyveromyces lactis and analyses of the nucleoid proteins

Mitochondrial (mt) nucleoids were isolated from yeast Kluyveromyces lactis with morphological intactness. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed more than 20 proteins that are associated with the mt-nucleoids. However, the protein profile of the mt-nucleoids of K. lactis was significantly different from that of the mt-nucleoid proteins from Saccharomyces cerevisiae. SDS-DNA PAGE, which detected an Abf2p, a major mitochondrial DNA-binding protein, among the mt-nucleoid proteins of S. cerevisiae on a gel, detected only a 17-kDa protein in the K. lactis mt-nucleoid proteins. The 17-kDa protein was purified as homogeneous from the mt-nucleoids by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography. The 17-kDa protein introduced a negative supercoil into circular plasmid DNA in the presence of topoisomerase I, as does S. cerevisiae Abf2p, and it packed K. lactis mtDNA into nucleoid-like particles in vitro. These results, together with the determination of the N-terminal amino acid sequence, suggested that the 17-kDa protein is an Abf2p homologue of K. lactis and plays structural roles in compacting mtDNA in cooperation with other nucleoid proteins.     

The modulation of the biological activities of mitochondrial histone Abf2p by yeast PKA and its possible role in the regulation of mitochondrial DNA content during glucose repression.

The mitochondrial histone, Abf2p, of Saccharomyces cerevisiae is essential for the maintenance of mitochondrial DNA (mtDNA) and appears to play an important role in the recombination and copy number determination of mtDNA. Abf2p, encoded by a nuclear gene, is a member of HMG1 DNA-binding protein family and has two HMG1-Box domains, HMG1-Box A and B. To investigate the role of Abf2p in the control of mtDNA copy number, we asked if the in vivo functions of Abf2p are regulated by the possible modification such as phosphorylation. We found that the N-terminal extended segment (KRPT(21)S(22)) of HMG1-Box A is rapidly and specifically phosphorylated by cAMP-dependent protein kinase (PKA) in vitro. The phosphorylation in this region inhibits the binding of Abf2p to all kinds of DNA including four-way junction DNA and the supercoiling activity of Abf2p itself. The abf2 mutant cells with an abf2(T21A/S22A) allele defective in the phosphorylation site have a severe defect in the regulation of mtDNA content during glucose repression in vivo. These observations suggest that the phosphorylation via PKA, that is activated during glucose repression, may regulate the in vivo functions of Abf2p for the control of mtDNA content during shift from gluconeogenic to fermentative growth.     

The mitochondrial genome. The nucleoid

Mitochondrial DNA (mtDNA) in cells is organized in nucleoids containing DNA and various proteins. This review discusses questions of organization and structural dynamics of nucleoids as well as their protein components. The structures of mt-nucleoid from different organisms are compared. The currently accepted model of nucleoid organization is described and questions needing answers for better understanding of the fine mechanisms of the mitochondrial genetic apparatus functioning are discussed.     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

Preparation of a Monoclonal Antibody Specific for a 48-kDa Protein from Mitochondrial Nucleoids of the Yeast, Saccharomyces cerevisiae

Monoclonal antibodies (mAbs) were raised against yeast mitochondrialnucleoids (mtnucleoids). In an analysis by a combination ofimmunofluorescence microscopy and staining with 4',6-diamidino-2-phenylindole(DAPI), one of them, designated YMN-1, distinctly stained mtnucleoids,which were visible as dots, in spheroplasts and in isolatedmitochondria. However, staining of isolated mt-nucleoids wasrather weak. YMN-1 mAb recognized a 48-kDa protein in immunoblotsof both mitochondrial and mt-nucleoid proteins. The 48-kDa proteinwas a minor component of mt-nucleoid proteins and was separatedfrom extract of both mitochondria and mt-nucleoids by immunoamnitychromatography. The affinity-purified 48-kDa protein reassociatedwith mt-nucleoids when mixed with isolated mt-nucleoids, asmonitored by immunofluorescence microscopy. The results suggestthat a large amount of 48-kDa protein is associated with mt-nucleoidsin vivo, and that lysis of mitochondria by the treatment withdetergent releases a considerable amount of this protein frommt-nucleoids during the isolation of mt-nucleoids. (Received June 25, 1992; Accepted November 16, 1992)     

A 22kDa protein specific for yeast mitochondrial nucleoids is an unidentified putative ribosomal protein encoded in open reading frame YGL068W

Summary. Mitochondrial-nucleoid (mt-nucleoid) proteins of the yeast Saccharomyces cerevisiae were separated by two-dimensional gel electrophoresis. Analysis of the N-terminal amino acid sequence showed that a 22kDa protein which is unique in the mt-nucleoid fraction is an unidentified protein encoded in the open reading frame YGL068W and shows a homology with the ribosomal protein L7/L12 of bacteria. We named this protein Mnp1p (for the mitochondrial-nucleoid protein 1). Immunoblotting of each fraction with an anti-Mnp1p antibody during the mt-nucleoid isolation showed that Mnp1p is highly concentrated in the mt-nucleoid fraction. Immunofluorescence microscopy suggested that Mnp1p is localized to mitochondria in vivo, and a significant amount of Mnp1p is associated with the mt-nucleoids. On the other hand, Northern blotting showed that a large amount of large and small mitochondrial ribosomal RNAs was not associated with the mt-nucleoids and remained in the supernatant after the isolation of mt-nucleoids. The null mutation of MNP1 led to a respiratory-deficient phenotype, but the morphology of the mt-nucleoids in the transformants carrying the null mutation was normal. These results suggest that a significant amount of Mnp1p plays a role as a major component of the mt-nucleoids.Correspondence and reprints: Department of Physics, Informatics, and Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan.     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

Analysis of mitochondrial DNA nucleoids in wild-type and a mutant strain of Saccharomyces cerevisiae that lacks the mitochondrial HMG box protein Abf2p.

DNA-protein complexes (nucleoids) are believed to be the segregating unit of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. A mitochondrial HMG box protein, Abf2p, is needed for maintenance of mtDNA in cells grown on rich dextrose medium, but is dispensible in glycerol grown cells. As visualized by 4',6'-diamino-2-phenylindole staining, mtDNA nucleoids in mutant cells lacking Abf2p ( delta abf2) are diffuse compared with those in wild-type cells. We have isolated mtDNA nucleoids and characterized two mtDNA-protein complexes, termed NCLDp-2 and NCLDs-2, containing distinct but overlapping sets of polypeptides. This protocol yields similar nucleoid complexes from the delta abf2 mutant, although several proteins appear lacking from NCLDs-2. Segments of mtDNA detected with probes to COXII, VAR1 and ori5 sequences are equally sensitive to DNase I digestion in NCLDs-2 and NCLDp-2 from wild-type cells and from the delta abf2 mutant. However, COXII and VAR1 sequences are 4-to 5-fold more sensitive to DNase I digestion of mtDNA in toluene-permeabilized mitochondria from the delta abf2 mutant than from wild-type cells, but no difference in DNase I sensitivity was detected with the ori5 probe. These results provide a first indication that Abf2p influences differential organization of mtDNA sequences.     

Phosphorylation of high-mobility-group proteins by the calcium-phospholipid-dependent protein kinase and the cyclic AMP-dependent protein kinase

Purified lamb thymus high-mobility-group (HMG) proteins 1, 2, and 17 have been investigated as potential substrates for the Ca2+-phospholipid-dependent protein kinase and the cAMP-dependent protein kinase. HMG proteins 1, 2, and 17 are phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the reactions are totally Ca2+ and lipid dependent and are not inhibited by the inhibitor protein of the cAMP-dependent protein kinase. HMG 17 is phosphorylated predominantly in a single seryl residue, Ser 24 in the sequence Gln-Arg-Arg-Ser 24-Ala-Arg-Leu-Ser 28-Ala-Lys, with the second seryl moiety, Ser 28, modified to a markedly lesser degree. HMGs 1 and 2 are also phosphorylated in only seryl residues but with each there are multiple phosphorylation sites. HMG 17, but not HMG 1 or 2, is also phosphorylated by the cAMP-dependent protein kinase with the site phosphorylated being the minor of the two phosphorylated by the Ca2+-phospholipid-dependent protein kinase; the Km for phosphorylation by the cAMP-dependent enzyme is 50-fold higher than that by the Ca2+-phospholipid-dependent enzyme. HMG 17 is an equally effective substrate for the Ca2+-phospholipid-dependent protein kinase either as the pure protein or bound to nucleosomes. Preliminary evidence has indicated that lamb thymus HMG 14 is also a substrate for the Ca2+-phospholipid-dependent enzyme. It is phosphorylated with a Km similar to that of HMG 17 (4-6 microM), and a comparison of tryptic peptides suggests that it is phosphorylated in a site that is homologous with Ser 24 of HMG 17 and distinct from the sites phosphorylated by the cAMP-dependent protein kinase.     

Isolation and Characterization of Mitochondrial Nucleoids from the Yeast Pichia jadinii

Mitochondrial (mt) nucleoids were isolated with a high degreeof purity from the yeast Pichia jadinii, in which the mitochondrialDNA (mtDNA) is linear. Field-inversion gel electrophoresis (FIGE)revealed that significant amounts of mtDNA could be isolatedintact, as linear molecules of 41 kbp, from the isolated mt-nucleoids.Fifteen different proteins were detected in the mt-nucleoidfraction and, eight of these proteins bound to DNA. The patternsof mt-nucleoid proteins and of the DNA-binding proteins aftergel electrophoresis in the presence of SDS were somewhat differentfrom those of such proteins from Saccharomyces cerevisiae. Thecorresponding proteins isolated from the mt-nucleoids of fourother species of yeast in the genera Pichia and Williopsis alsodiffered from one another in terms of electrophoretic mobilityin the presence of SDS. In immunoblotting experiments, antibodiesthat had been raised against the 67-kDa protein of mt-nucleoidsfrom S. cerevisiae and the YMN-1 monoclonal antibody that isspecific for a 48-kDa protein in the mt-nucleoids from S. cerevisiaedid not recognize any proteins in the mt-nucleoids from Pichiajadinii and four other species of yeast. The results suggestthe considerable diversity of the proteins in the mt-nucleoidsof yeasts. (Received March 28, 1996; Accepted June 19, 1996)     

Unwinding of DNA by nonhistone protein HMG1 and HMG2

The enzyme kinetic studies with endonucleases specific for single-stranded DNA and the thermal denaturation analyses of DNA showed that a high mobility group (HMG) nonhistone protein fraction HMG (1 + 2), composed of HMG1 and HMG2, has an activity to unwind DNA partially at low protein-to-DNA weight ratio. Isolated HMG1 and HMG2 have the same activity. Divalent cations such as Mg++ or Ca++ were necessary for the unwinding reaction. A peptide containing high glutamic and aspartic (HGA) region, isolated from the tryptic digest of HMG (1 + 2), unwound DNA depending on the presence of Mg++ or Ca++, suggesting that the HMA region in HMG protein is the active site for the DNA unwinding reaction. Poly-L-glutamic acid, employed as a model peptide of the HGA region, showed the activity. Finally, mechanisms of the DNA unwinding reaction by the HMG protein and possible role of the divalent cations are discussed.     

Evolutionary tinkering with mitochondrial nucleoids

Mitochondrial DNA (mtDNA) is organized in nucleoprotein particles called nucleoids. Each nucleoid, which is considered a heritable unit of mtDNA, might contain several copies of the mitochondrial genome and several different proteins. Some nucleoid-associated proteins, such as the high mobility group (HMG) box family, have well defined functions in mtDNA maintenance and packaging; others, such as Aco1 and IIv5, are bifunctional, fulfilling their roles in nucleoids in addition to well established metabolic functions. The fact that the HMG box mtDNA packaging proteins are of eukaryotic rather than bacterial origin and also that every organism seems to have a unique set of nucleoid-associated proteins suggests that evolutionary tinkering occurred to reinvent mitochondrial nucleoprotein during the evolution of mitochondrial genomes.     

Does high-mobility-group non-histone protein HMG 1 interact specifically with histone H1 subfractions?

The interaction of the non-histone chromosomal protein HMG (high-mobility group) 1 with histone H1 subfractions was investigated by equilibrium sedimentation and n.m.r. sectroscopy. In contrast with a previous report [Smerdon & Isenberg (1976) Biochemistry 15, 4242--4247], it was found, by using equilibrium-sedimentation analysis, that protein HMG 1 binds to all three histone H1 subfractions CTL1, CTL2, and CTL3, arguing against there being a specific interaction between protein HMG 1 and only two of the subfractions, CTL1 and CTL2. Raising the ionic strength of the solutions prevents binding of protein HMG 1 to total histone H1 and the three subfractions, suggesting that the binding in vitro is simply a non-specific ionic interaction between acidic regions of the non-histone protein and the basic regions of the histone. Protein HMG 1 binds to histone H5 also, supporting this view. The above conclusions are supported by n.m.r. studies of protein HMG 1/histone H1 subfraction mixtures. When the two proteins were mixed, there was little perturbation of the n.m.r. spectra and there was no evidence for specific interaction of protein HMG 1 with any of the subfractions. It therefore remains an open question as to whether protein HMG 1 and histone H1 are complexed together in chromatin.     

Abundance of mRNAs encoding HMG1/HMG2 class high-mobility-group DNA-binding proteins are differentially regulated in cotyledons of Pharbitis nil

The abundance of an mRNA encoding an HMG1/2 protein from Pharbitis nil (HMG1) has been previously shown to be regulated by light and an endogenous rhythm in cotyledons. A second Pharbitis nil HMG cDNA (HMG2) was characterized. The sequence of HMG2 was 82% and 86% identical to HMG1 at the nucleotide and amino acid level, respectively. As with HMG1, HMG2 mRNA was detected in all vegetative tissues and was most abundant in roots. However, unlike HMG1, HMG2 mRNA abundance did not increase upon transfer of cotyledons to darkness and did not exhibit regulation by an endogenous circadian rhythm when maintained in continuous darkness over a 68 h period. Similarly, while the abundance of HMG1 mRNA during a dark period that induced photoperiodically controlled flowering was dramatically affected by brief light exposure (night break), this treatment had no effect on HMG2 mRNA abundance. Collectively, these data are consistent with a role of HMG1 in contributing to the circadian-regulated and/or dark-regulated gene expression with constitutive expression of HMG2 playing a housekeeping role in the general regulation of gene expression in Pharbitis nil cotyledons.