Isamu Miyakawa

Meiotic karyotype of the yeast Saccharomyces cerevisiae

A cytogenetic study of the meiotic chromosomes of the budding yeast Saccharomyces cerevisiae was undertaken by high resolution epifluorescence microscopy. Condensation of chromatin into separate chromosomes takes place during prophase I. At metaphase I, there are 16 separate and distinct bivalents which are roughly classified into three groups by morphological differences and DNA content.

Identification of the YMN-1 antigen protein and biochemical analyses of protein components in the mitochondrial nucleoid fraction of the yeast Saccharomyces cerevisiae

Summary. We analyzed the protein components contained in the mitochondrial nucleoid (mt-nucleoid) fraction of the yeast Saccharomyces cerevisiae. Immunoblotting with anti-Abf2p antibody demonstrated the association of Abf2p, a major mitochondrial DNA-binding protein, with the mt-nucleoids. In contrast, porin and cytochrome c oxidase subunit III (CoxIIIp) were not detected by immunoblotting in the mt-nucleoid fraction. The YMN-1 monoclonal antibody recognized a 48 kDa protein of the mt-nucleoid fraction. The N-terminal amino acid sequence of the protein and immunological evidence showed that the YMN-1 monoclonal antibody recognizes dihydrolipoyl transsuccinylase (KE2), which is one of the constituents of the α-ketoglutarate dehydrogenase complex (KGDC). α-Ketoglutarate dehydrogenase (KE1) and dihydrolipoyl dehydrogenase (E3), which are other subunits of KGDC, were also detected in the mt-nucleoid fraction. An enzyme assay of the mt-nucleoid fraction showed that cytochrome c oxidase and fumarase activity were barely detected in the fraction, but the specific activity of KGDC in the mt-nucleoid fraction was relatively high and was approximately 60% of the specific activity in the mitochondrial fraction. Three components of KGDC were detected in the DNA-binding protein fractions after DNA-cellulose column chromatography of mt-nucleoid proteins. These results suggested that a part of KGDC in the mitochondrial matrix is associated with mt-nucleoids in vivo.

Mitochondrial nucleoids from the yeast Candida parapsilosis: expansion of the repertoire of proteins associated with mitochondrial DNA.

Molecules of mitochondrial DNA (mtDNA) are packed into nucleic acid-protein complexes termed mitochondrial nucleoids (mt-nucleoids). In this study, we analysed mt-nucleoids of the yeast Candida parapsilosis, which harbours a linear form of the mitochondrial genome. To identify conserved as well as specific features of mt-nucleoids in this species, we employed two strategies for analysis of their components. First, we investigated the protein composition of mt-nucleoids isolated from C. parapsilosis mitochondria, determined N-terminal amino acid sequences of 14 proteins associated with the mt-nucleoids and identified corresponding genes. Next, we complemented the list of mt-nucleoid components with additional candidates identified in the complete genome sequence of C. parapsilosis as homologues of Saccharomyces cerevisiae mt-nucleoid proteins. Our approach revealed several known mt-nucleoid proteins as well as additional components that expand the repertoire of proteins associated with these cytological structures. In particular, we identified and purified the protein Gcf1, which is abundant in the mt-nucleoids and exhibits structural features in common with the mtDNA packaging protein Abf2 from S. cerevisiae. We demonstrate that Gcf1p co-localizes with mtDNA, has DNA-binding activity in vitro, and is able to stabilize mtDNA in the S. cerevisiae Deltaabf2 mutant, all of which points to a role in the maintenance of the C. parapsilosis mitochondrial genome. Importantly, in contrast to Abf2p, in silico analysis of Gcf1p predicted the presence of a coiled-coil domain and a single high-mobility group (HMG) box, suggesting that it represents a novel type of mitochondrial HMG protein.

Isolation of giant mitochondrial nucleoids from the yeastSaccharomyces cerevisiae

SummaryThe yeast cellsSaccharomyces cerevisiae grown up to stationary phase under either anaerobic conditions, or aerobic conditions in the presence of a respiratory inhibitor, antimycin A, had distinctive giant mitochondrial nucleoids (mt-nucleoids) (apparent diameter 0.6–0.9 μm) in contrast with the small mt-nucleoids (apparent diameter 0.2–0.4 μm) in respiratory-sufficient cells grown aerobically, as revealed by DAPI-fluorescence microscopy. The cytoplasmic respiratory-deficient cells (rho− cells), which were induced by treatment of wild-type cells with ethidium bromide, showed both giant and small mt-nucleoids of irregular size. In order to examine the structural and functional differences between giant and small mt-nucleoids, the former were successfully isolated from spheroplasts of three different cells by differential centrifugation and centrifugation on a discontinuous sucrose gradient. The isolated giant mt-nucleoids were intact in the morphology and were free of significant contamination by nuclear chromatin. The number of protein components involved in each of three different giant mt-nucleoids was similar to the number in small mt-nucleoids from aerobically grown cells, though a few noticeable differences were also recognized. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 26 kDa, and 20 kDa were the main components of small mt-nucleoids from aerobically grown cells as detected by chromatography on native DNA-cellulose. In contrast, the 67 kDa and 52 kDa proteins were hardly detected in corresponding fractions of giant mt-nucleoids from anaerobically grown cells and from rho− cells grown aerobically. On the other hand, mt-nucleoids from aerobically grown cells in the presence of antimycin A seemed to lack the 67 kDa protein but to have a small amount of the 52 kDa protein. This is the first demonstration of the variance of protein species involved in yeast mt-nucleoids according to the respiratory activity of mitochondria.

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.

Behavior of mitochondria, microtubules, and actin in the triangular yeastTrigonopsis variabilis

Dimorphic yeastTrigonopsis variabilis is a unique species that can form either an ellipsoidal or a triangular cell depending upon nutritional conditions. This fluorescence microscopic study was intended to correlate morphological changes of mitochondria in the triangular cells with the distribution of the cytoskeleton. In addition, unique features in the behavior of the cytoskeleton were also examined during triangular cell formation. In log-phase cells stained with 4′,6-diamidino-2-phenylindole, mitochondrial nucleoids appeared as a string of beads throughout the vegetative growth. The profile of mitochondria stained by 3,3′-dihexyloxacarbocyanine iodide showed a network corresponding to the fluorescence images of mitochondrial nucleoids in both mother and daughter cells. Cell-cycle-dependent fragmentation of mitochondria was not discerned. As the culture reached stationary phase, a network of mitochondria gradually changed to form unique rings that were located near the angles of triangular cells. When examined by immunofluorescence microscopy with anti-tubulin antibody, microtubules were found to be well developed along the sides of cells in the cytoplasm ofT. variabilis interphase cells. Although distributions of microtubules and mitochondria are different during cell cycle as a whole, cytoplasmic microtubules frequently extended along a part of the mitochondria in budded cells, suggesting correlation of microtubules and mitochondria. Rhodamine-phalloidin staining revealed both actin patches and cables. Actin cables elongated from mother cells into the buds and showed close proximity to mitochondria, although complete overlapping of both structures was rare. Moreover, actin patches localized on the mitochondrial network at a frequency of 65%. These results suggested that actin cables and patches, as well as microtubules, participated in the distribution of mitochondria. The localization of actin patches separated towards opposite ends at a bud tip when the bud grew to medium size. The unique localization of actin patches is responsible for bi-directional growth of the bud, forming triangular cells.

Organization and dynamics of yeast mitochondrial nucleoids

Mitochondrial DNA (mtDNA) is packaged by association with specific proteins in compact DNA-protein complexes named mitochondrial nucleoids (mt-nucleoids). The budding yeast Saccharomyces cerevisiae is able to grow either aerobically or anaerobically. Due to this characteristic, S. cerevisiae has been extensively used as a model organism to study genetics, morphology and biochemistry of mitochondria for a long time. Mitochondria of S. cerevisiae frequently fuse and divide, and perform dynamic morphological changes depending on the culture conditions and the stage of life cycle of the yeast cells. The mt-nucleoids also dynamically change their morphology, accompanying morphological changes of mitochondria. The mt-nucleoids have been isolated morphologically intact and functional analyses of mt-nucleoid proteins have been extensively performed. These studies have revealed that the functions of mt-nucleoid proteins are essential for maintenance of mtDNA. The aims of this review are to summarize the history on the research of yeast mt-nucleoids as well as recent findings on the organization of the mt-nucleoids and mitochondrial dynamics.

Morphology of mitochondrial nucleoids in respiratory-deficient yeast cells varies depending on the unit length of the mitochondrial DNA sequence.

We investigated the morphology of mitochondrial nucleoids (mt-nucleoids) and mitochondria in Saccharomyces cerevisiae rho(+) and rho(-) cells with DAPI staining and mitochondria-targeted GFP. Whereas the mt-nucleoids appeared as strings of beads in wild-type rho(+) cells at log phase, the mt-nucleoids in hypersuppressive rho(-) cells (HS40 rho(-) cells) appeared as distinct punctate structures. In order to elucidate whether the punctate mt-nucleoids are common to other rho(-) cells, we observed the mt-nucleoids in rho(-) strains that retain different unit lengths of the mitochondrial DNA (mtDNA) sequence. As a result, rho(-) cells that have long mtDNA sequences, of more than 30 kb, had mt-nucleoids with a strings-of-beads appearance in tubular mitochondria. In contrast, rho(-) cells that have short mtDNA sequences, of <1 kb, had punctate mt-nucleoids in tubular mitochondria. This indicates that the morphology of mt-nucleoids in rho(-) cells significantly varies depending on the unit length of their mtDNA sequence. Analyses of mt-nucleoids suggest that the punctate mt-nucleoids in HS40 rho(-) cells consist of concatemeric mtDNAs and oligomeric circular mtDNAs associated with Abf2p and other nucleoid proteins.

Tellurium as a valuable tool for studying the prokaryotic origins of mitochondria

Mitochondria are eukaryotic organelles which contain the own genetic material and evolved from free-living Eubacteria, namely hydrogen-producing Alphaproteobacteria. Since 1965, biologists provided, by research at molecular level, evidence for the prokaryotic origins of mitochondria. However, determining the precise origins of mitochondria is challenging due to inherent difficulties in phylogenetically reconstructing ancient evolutionary events. The use of new tools to evidence the prokaryotic origin of mitochondria could be useful to gain an insight into the bacterial endosymbiotic event that resulted in the permanent acquisition of bacteria, from the ancestral cell, that through time were transformed into mitochondria. Electron microscopy has shown that both proteobacterial and yeast cells during their growth in the presence of increasing amount of tellurite resulted in dose-dependent blackening of the culture due to elemental tellurium (Te(0)) that formed large deposits either along the proteobacterial membrane or along the yeast cell wall and mitochondria. Since the mitochondrial inner membrane composition is similar to that of proteobacterial membrane, in the present work we evidenced the black tellurium deposits on both, cell wall and mitochondria of ρ(+) and respiratory deficient ρ(-) mutants of yeast. A possible role of tellurite in studying the evolutionary origins of mitochondria will be discussed.

Correction for Bakkaiova et al., The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

Volume 13, no. 9, p. [1143–1157][1], 2014: Based on our recent correspondence with Claude Gaillardin (AgroParisTech, Jouy-en-Josas, France), we want to modify our proposition about translation initiation of YlMHB1 presented in our recent paper (doi:[10.1128/EC.00092-14][2]). In the original report