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Primary plastids originated from a free-living cyanobacterial ancestor and possess their own genomes-probably a few DNA copies. These genomes, which are organized in centrally located plastid nuclei (CN-type pt-nuclei), are produced from preexisting plastids by binary division. Ancestral algae with a CN-type pt-nucleus diverged and evolved into two basal eukaryotic lineages: red algae with circular (CL-type) pt-nuclei and green algae with scattered small (SN-type) pt-nuclei. Although the molecular dynamics of pt-nuclei in green algae and plants are now being analyzed, the process of the conversion of the original algae with a CN-type pt-nucleus to red algae with a CL-type one has not been studied. Here, we show that the CN-type pt-nucleus in the primitive red alga Cyanidioschyzon merolae can be changed to the CL-type by application of drying to produce slight cell swelling. This result implies that CN-type pt-nuclei are produced by compact packing of CL-type ones, which suggests that a C. merolae-like alga was the original progenitor of the red algal lineage. We also observed that the CL-type pt-nucleus has a chain-linked bead-like structure. Each bead is most likely a small unit of DNA, similar to CL-type pt-nuclei in brown algae. Our results thus suggest a C. merolae-like alga as the candidate for the secondary endosymbiont of brown algae.

Here we report the complete organellar genome sequences of Medakamo hakoo, a green alga identified in freshwater in Japan. It has 90.8-kb plastid and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein coding genes, respectively, representing the smallest organellar genome among currently known core Trebouxiophyceae.

Mitochondria, which evolved from a free-living bacterial ancestor, contain their own genomes and genetic systems and are produced from preexisting mitochondria by binary division. The mitochondrion-dividing (MD) ring is the main skeletal structure of the mitochondrial division machinery. However, the assembly mechanism and molecular identity of the MD ring are unknown. Multi-omics analysis of isolated mitochondrial division machinery from the unicellular alga Cyanidioschyzon merolae revealed an uncharacterized glycosyltransferase, MITOCHONDRION-DIVIDING RING1 (MDR1), which is specifically expressed during mitochondrial division and forms a single ring at the mitochondrial division site. Nanoscale imaging using immunoelectron microscopy and componential analysis demonstrated that MDR1 is involved in MD ring formation and that the MD ring filaments are composed of glycosylated MDR1 and polymeric glucose nanofilaments. Down-regulation of MDR1 strongly interrupted mitochondrial division and obstructed MD ring assembly. Taken together, our results suggest that MDR1 mediates the synthesis of polyglucan nanofilaments that assemble to form the MD ring. Given that a homolog of MDR1 performs similar functions in chloroplast division, the establishment of MDR1 family proteins appears to have been a singular, crucial event for the emergence of endosymbiotic organelles.

In many eukaryotes, cytokinesis proceeds in two successive steps: first, ingression of the cleavage furrow and second, abscission of the intercellular bridge. In animal cells, the actomyosin contractile ring is involved in the first step, while the endosomal sorting complex required for transport (ESCRT), which participates in various membrane fusion/fission events, mediates the second step. Intriguingly, in archaea, ESCRT is involved in cytokinesis, raising the hypothesis that the function of ESCRT in eukaryotic cytokinesis descended from the archaeal ancestor. In eukaryotes other than in animals, the roles of ESCRT in cytokinesis are poorly understood. To explore the primordial core mechanisms for eukaryotic cytokinesis, we investigated ESCRT functions in the unicellular red alga Cyanidioschyzon merolae that diverged early in eukaryotic evolution. C. merolae provides an excellent experimental system. The cell has a simple organelle composition. The genome (16.5 Mb, 5335 genes) has been completely sequenced, transformation methods are established, and the cell cycle is synchronized by a light and dark cycle. Similar to animal and fungal cells, C. merolae cells divide by furrowing at the division site followed by abscission of the intercellular bridge. However, they lack an actomyosin contractile ring. The proteins that comprise ESCRT-I–IV, the four subcomplexes of ESCRT, are partially conserved in C. merolae. Immunofluorescence of native or tagged proteins localized the homologs of the five ESCRT-III components [charged multivesicular body protein (CHMP) 1, 2, and 4–6], apoptosis-linked gene-2-interacting protein X (ALIX), the ESCRT-III adapter, and the main ESCRT-IV player vacuolar protein sorting (VPS) 4, to the intercellular bridge. In addition, ALIX was enriched around the cleavage furrow early in cytokinesis. When the ESCRT function was perturbed by expressing dominant-negative VPS4, cells with an elongated intercellular bridge accumulated—a phenotype resulting from abscission failure. Our results show that ESCRT mediates cytokinetic abscission in C. merolae. The fact that ESCRT plays a role in cytokinesis in archaea, animals, and early diverged alga C. merolae supports the hypothesis that the function of ESCRT in cytokinesis descended from archaea to a common ancestor of eukaryotes.

Cells of the red alga Cyanidioschyzon merolae contain similar types of organelles as other model eukaryotic organisms (see Chap. 2). However, C. merolae cells are distinct in that they contain fewer organelles, with only one nucleus, mitochondrion, chloroplast, peroxisome, Golgi, and endoplasmic reticulum (ER) per cell, and only a few lysosomes or vacuoles. C. merolae cells lack a rigid cell wall, and the plasma membrane is thick and elastic. These biological features facilitate the isolation of distinct types of organelles from C. merolae cells. Standardized protocols have been established to isolate organelles with bilayer membranes (e.g., chloroplasts and mitochondria) and single-layer membranes (e.g., peroxisomes and lysosomes). The isolated organelles can be used to address biological questions by performing in vitro experiments and biochemical analyses of purified target molecules. Together with imaging and genome information analyses, the resulting data will provide insights into key components involved in fundamental cellular mechanisms. In this chapter, we provide detailed protocols for the isolation of specific organelles from C. merolae cells.

The Golgi body has important roles in modifying, sorting, and transport of proteins and lipids. Eukaryotic cells have evolved in various ways to inherit the Golgi body from mother to daughter cells, which allows the cells to function properly immediately after mitosis. Here we used Cyanidioschyzon merolae, one of the most suitable systems for studies of organelle dynamics, to investigate the inheritance of the Golgi. Two proteins, Sed5 and Got1, were used as Golgi markers. Using immunofluorescence microscopy, we demonstrated that C. merolae contains one to two Golgi bodies per cell. The Golgi body was localized to the perinuclear region during the G1 and S phases and next to the spindle poles in a microtubule-dependent manner during M phase. It was inherited together with spindle poles upon cytokinesis. These observations suggested that Golgi inheritance is dependent on microtubules in C. merolae.

Endoplasmic reticulum (ER) is a major site for secretory protein folding and lipid synthesis. Since ER cannot be synthesized de novo, it must be inherited during the cell cycle. Studying ER inheritance can however be difficult because the ER of typical plant and animal cells is morphologically complex. Therefore, our study used Cyanidioschyzon merolae, a species that has a simple ER structure, to investigate the inheritance of this organelle. Using immunofluorescence microscopy, we demonstrated that C. merolae contains a nuclear ER (nuclear envelope) and a small amount of peripheral ER extending from the nuclear ER. During mitosis, the nuclear ER became dumbbell-shaped and underwent division. Peripheral ER formed ring-like structures during the G1 and S phases, and extended toward the mitochondria and cell division planes during the M phase. These observations indicated that C. merolae undergoes closed mitosis, whereby the nuclear ER does not diffuse, and the peripheral ER contains cell cycle-specific structures.

It is generally believed that the cell cycle consists essentially of the mitotic cycle, which involves mitosis and cytokinesis. These processes are becoming increasingly well understood at the molecular level. However, successful cell reproduction requires duplication and segregation (inheritance) of all of the cellular contents, including not only the cell-nuclear genome but also intracellular organelles. Eukaryotic cells contain at least three types of double membrane-bounded organelles (cell nucleus, mitochondria and plastids), four types of single membrane-bounded organelles (endoplasmic reticulum, Golgi apparatus, lysosomes and microbodies) and the cytoskeleton, which comprises tubulin-based structures (including microtubules, centrosome and spindle) and actin microfilaments. These membrane-bounded organelles cannot be formed de novo and daughter organelles must be inherited from parent organelles during cell cycle. Regulation of organelle division and its coordination with the progression of the cell cycle involves a sequence of events that are subjected to precise spatio-temporal control. Considering that the cells of higher animals and plants contain many organelles which tend to behave somewhat randomly, there is little information concerning the division and inheritance of these double- and single-membrane-bounded organelles during the cell cycle. Here, we summarize the current cytological and morphological knowledge of the cell cycle, including the division cycles of seven membrane-bounded and some non-membrane-bounded organelles. The underlying mechanisms and the biological relevance of these processes are discussed, particularly with respect to cells of the primitive alga Cyanidioschyzon merolae that have a minimum of organelles. We discuss unsolved problems and future perspectives opened by recent studies.

Chloroplast Division Machinery The machinery for photosynthesis, which captures the Sun's energy to generate carbohydrates, generally resides in subcellular chloroplasts of plant cells. Chloroplasts must divide as the plant cell divides, but to do so requires their own plastid dividing machinery. Yoshida et al. (p. 949 : see the cover) have now analyzed the plastid dividing machinery of the single-celled alga Cyanidioschyzon merolae , whose cells each contain a single chloroplast. The plastid dividing machinery is made up of polysaccharide chains and the proteins that make them, which together generate a ring that constricts to physically divide the chloroplast.

Vacuoles/lysosomes function in endocytosis and in storage and digestion of metabolites. These organelles are inherited by the daughter cells in eukaryotes. However, the mechanisms of this inheritance are poorly understood because the cells contain multiple vacuoles that behave randomly. The primitive red alga Cyanidioschyzon merolae has a minimum set of organelles. Here, we show that C. merolae contains about four vacuoles that are distributed equally between the daughter cells by binding to dividing mitochondria. Binding is mediated by VIG1, a 30-kD coiled-coil protein identified by microarray analyses and immunological assays. VIG1 appears on the surface of free vacuoles in the cytosol and then tethers the vacuoles to the mitochondria. The vacuoles are released from the mitochondrion in the daughter cells following VIG1 digestion. Suppression of VIG1 by antisense RNA disrupted the migration of vacuoles. Thus, VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance in C. merolae.

Vacuoles function in endocytosis, storage and digestion of metabolites in eukaryotic cells. They are inherited by the daughter cells. However, the mechanisms of vacuole inheritance are poorly understood because the cells contain multiple vacuoles that behave randomly. Cyanidioschyzon merolae cell has a minimum set of organelles. The vacuoles were equally inherited by the daughter cells by binding to dividing mitochondria. The binding was mediated by VIG1. However, the role of the binding in the vacuoles inheritance was poorly understood. We examined it by inhibiting the binding cytochemically. The vacuoles, which were not bound to mitochondria, were not equally inherited by the daughter cells. As the results, vacuole-less daughter cells were generated. These results suggested that the binding contributed the equal inheritance of vacuoles and ensured the permanence of vacuoles in daughter cells.

Plant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate-rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF-MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density-gradient ultracentrifugation. The vacuole-containing fraction was subjected to SDS-PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non-lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o-methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin-tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features.

Previous cell cycle studies have been based on cell-nuclear proliferation only. Eukaryotic cells, however, have double membranes-bound organelles, such as the cell nucleus, mitochondrion, plastids and single-membrane-bound organelles such as ER, the Golgi body, vacuoles (lysosomes) and microbodies. Organelle proliferations, which are very important for cell functions, are poorly understood. To clarify this, we performed a microarray analysis during the cell cycle of Cyanidioschyzon merolae. C. merolae cells contain a minimum set of organelles that divide synchronously. The nuclear, mitochondrial and plastid genomes were completely sequenced. The results showed that, of 158 genes induced during the S or G2-M phase, 93 were known and contained genes related to mitochondrial division, ftsZ1-1, ftsz1-2 and mda1, and plastid division, ftsZ2-1, ftsZ2-2 and cmdnm2. Moreover, three genes, involved in vesicle trafficking between the single-membrane organelles such as vps29 and the Rab family protein, were identified and might be related to partitioning of single-membrane-bound organelles. In other genes, 46 were hypothetical and 19 were hypothetical conserved. The possibility of finding novel organelle division genes from hypothetical and hypothetical conserved genes in the S and G2-M expression groups is discussed.

Cyanidioschyzon merolae is considered as a suitable model system for studies of organelle differentiation, proliferation and partitioning. Here, we have identified and characterized vacuoles in this organism and examined the partitioning of vacuoles using fluorescence and electron microscopy. Vacuoles were stained with the fluorescent aminopeptidase substrate 7-amino-4-chloromethylcoumarin L: -arginine amide, acidotrophic dyes quinacrine and LysoTracker, and 4',6-diamidino-2-phenyl indole, which, at a high concentration, stains polyphosphate. Vacuoles have been shown to be approximately 500 nm in diameter with a mean of around five per interphase cell. The vacuolar H(+)-ATPase inhibitor concanamycin A blocked the accumulation of quinacrine in the vacuoles, suggesting the presence of the enzyme on these membranes. Electron microscopy revealed that the vacuoles were single membrane-bound organelles with an electron-dense substance, often containing a thick layer surrounding the membrane. Immunoelectron microscopy using an anti-vacuolar-H(+)-pyrophosphatase antibody revealed the presence of the enzyme on these membranes. In interphase cells, vacuoles were distributed in the cytoplasm, while in mitotic cells they were localized adjacent to the mitochondria. Filamentous structures were observed between vacuoles and mitochondria. Vacuoles were distributed almost evenly to daughter cells and redistributed in the cytoplasm after cytokinesis. The change in localization of vacuoles also happened in microtubule-disrupted cells. Since no actin protein or filaments have been detected in C. merolae, this result suggests an intrinsic mechanism for the movement of vacuoles that differs from commonly known mechanisms mediated by microtubules and actin filaments.

Chloroplast division involves plastid-dividing, dynamin, and FtsZ (PDF) rings. We isolated intact supertwisted (or spiral) and circular PDF machineries from chloroplasts of the red alga Cyanidioschyzon merolae . After individual intact PDF machineries were stretched to four times their original lengths with optical tweezers, they spontaneously returned to their original sizes. Dynamin-released PDF machineries did not retain the spiral structure and could not be stretched. Thus, dynamin may generate the motive force for contraction by filament sliding in dividing chloroplasts, in addition to pinching-off the membranes.

The ultrasmall unicellular red alga Cyanidioschyzon merolae lives in the extreme environment of acidic hot springs and is thought to retain primitive features of cellular and genome organization. We determined the 16.5-Mb nuclear genome sequence of C. merolae 10D as the first complete algal genome. BLASTs and annotation results showed that C. merolae has a mixed gene repertoire of plants and animals, also implying a relationship with prokaryotes, although its photosynthetic components were comparable to other phototrophs. The unicellular green alga Chlamydomonas reinhardtii has been used as a model system for molecular biology research on, for example, photosynthesis, motility, and sexual reproduction. Though both algae are unicellular, the genome size, number of organelles, and surface structures are remarkably different. Here, we report the characteristics of double membrane- and single membrane-bound organelles and their related genes in C. merolae and conduct comparative analyses of predicted protein sequences encoded by the genomes of C. merolae and C. reinhardtii. We examine the predicted proteins of both algae by reciprocal BLASTP analysis, KOG assignment, and gene annotation. The results suggest that most core biological functions are carried out by orthologous proteins that occur in comparable numbers. Although the fundamental gene organizations resembled each other, the genes for organization of chromatin, cytoskeletal components, and flagellar movement remarkably increased in C. reinhardtii. Molecular phylogenetic analyses suggested that the tubulin is close to plant tubulin rather than that of animals and fungi. These results reflect the increase in genome size, the acquisition of complicated cellular structures, and kinematic devices in C. reinhardtii.

Cyanidioschyzon merolae is considered to be a suitable model system for cytologial studies of organelle proliferation and partitioning because these unicellular cells contain each organelle singly. However, lysosomes of C. merolae have yet to be identified. Polyphosphate have been known to be accumulated in the vacuoles of many microorganisms including alga. The cells stained with Neisser staining method, which visualizes polyphosphate bodies, showed the lysosome-like structures. They were about 500 nm in diameter and usually found as four copies in a single cell. The structures changed their localization dynamically during the cell cycle. During interphase, they were observed in the cytosol. At the beginning of mitosis, they moved over the mitochondria. During cytokinesis, they were inherited to the daughter cells almost evenly, suggesting the existence of mechanisms for the ordered partitioning.

In the mitochondria of primitive eukaryotes, FtsZ and dynamin are part of the machinery involved in division of the inner and outer membranes, respectively. These genes also commonly function in the same manner during chloroplast division. In this study, a relationship between the localization of the inner and outer division machinery was directly shown for the first time. Triple immunofluorescent labeling was performed in the red alga Cyanidioschyzon merolae by a device using narrow bandpass filter sets and bright photostable dyes. FtsZ (CmFtsZ1) and dynamin (CmDnm1) localizations were examined simultaneously throughout the mitochondrial division cycle with an alternative mitochondrial marker protein, the mitochondrial translation elongation factor EF-Tu, whose localization was also shown to be identical to the mitochondrial matrix. FtsZ and dynamin did not necessarily co-localize when both were recruited to the mitochondrial constriction site, indicating that inner and outer dividing machineries are not in tight association during the late stage of division. (J Histochem Cytochem 52:843–849, 2004)

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

Although the nuclear genome sequence of Cyanidioschyzon merolae 10D, a unicellular red alga, was recently determined, DNA transformation technology that is important as a model plant system has never been available thus far. In this study, improved culture conditions resulted in a faster growth rate of C. merolae in liquid medium (doubling time = 9.2 h), and colony formation on gellan gum plates. Using these conditions, spontaneous mutants (5-fluoroortic acid resistant) deficient in the UMP synthase gene were isolated. The lesions were then restored by introducing the wild-type UMP synthase gene into the cells suggesting DNA transformation by homologous recombination.

Cyanidioschyzon merolae is a unicellular red alga that lives in acidic hot springs. The genome sequence of C. merolae has been completely read, but a lack of transformation systems still limits its application in genetics. To choose an appropriate drug for use in selectable media, we examined the effects of seven antibiotics on the growth of C. merolae. Only cycloheximide, an inhibitor of protein synthesis, effectively inhibited the growth. We noticed that there was a population that could survive in the presence of cycloheximide and succeeded in isolating six cycloheximide-resistant clones. All these clones have the same single mutation in the ribosomal protein L29 gene that encodes a ribosomal protein.

To understand the regulatory mechanisms of chloroplast proliferation, chloroplast replication was studied in cultured leaf disks cut from plants of 25 species. In leaf disks from Brassica rapa var. perviridis, the number of chloroplasts per cell increased remarkably in culture. We examined chloroplast replication in this plant in vivo and in culture media with and without benzyladenine, a cytokinin. In whole plants, leaf cells undergo two phases from leaf emergence to full expansion: an early proliferative stage, in which mitosis occurs, and a differentiational stage after mitosis has diminished. During the proliferative stage, chloroplast replication keeps pace with cell division. In the differentiational phase, cell division ceases but chloroplast replication continues for two or three more cycles, with the number of chloroplasts per cell reaching about 60. In the leaf disks, the number of chloroplasts per cell increased from about 18 to 300 without benzyladenine, and to over 600 with benzyladenine, indicating that this cytokinin enhances chloroplast replication in cultured tissue. We also studied changes in ploidy and cell volume between in vivo cells and cells grown in culture with and without benzyladenine. Ploidy and cell volume increased in a manner very similar to that of the number of chloroplasts, suggesting a relationship between these phenomena.

Conserved domains of two types of LTR retrotransposons, Ty1–copia- and Ty3–gypsy-like retrotransposons, were isolated from the dioecious plant Silene latifolia, whose sex is determined by X and Y chromosomes. Southern hybridization analyses using these retrotransposons as probes resulted in identical patterns from male and female genomes. Fluorescence in situ hybridization indicated that these retrotransposons do not accumulate specifically in the sex chromosomes. These results suggest that recombination between the sex chromosomes of S. latifolia has not been severely reduced. Conserved reverse transcriptase regions of Ty1–copia-like retrotransposons were isolated from 13 different Silene species and classified into two major families. Their categorization suggests that parallel divergence of the Ty1–copia-like retrotransposons occurred during the differentiation of Silene species. Most functional retrotransposons from three dioecious species, S. latifolia, S. dioica, and S. diclinis, fell into two clusters. The evolutionary dynamics of retrotransposons implies that, in the genus Silene, dioecious species evolved recently from gynodioecious species.Key words: retrotransposon, dioecious plant, sex chromosome.

The ability of the primitive red alga Cyanidioschyzon merolae to adapt to high temperatures was utilized to produce thermotolerant transgenic plants. C. merolae inhabits an extreme environment (42 degrees C, pH 2.5) and the nuclear, mitochondrial, and plastid genomes have been sequenced. We analyzed expressed sequence tag (EST) data to reveal mechanisms of tolerance to high temperatures. The stromal ascorbate peroxidase (CmstAPX) that scavenges reactive oxygen species (ROS) was expressed at high levels (4th of 4,479 entries), thus, it offers clues to understanding high-temperature tolerance. CmstAPX has a chloroplast transit peptide (cTP) and a peroxidase domain. The peroxidase domain of CmstAPX has deletions and insertions when compared with that of Arabidopsis thaliana stromal APX (AtstAPX). To clarify aspects of tolerance to oxidative and high-temperature stress, we produced transgenic A. thaliana plants overexpressing CmstAPX and AtstAPX. CmstAPX plants showed higher activities of soluble APX than those of wild-type and AtstAPX plants. Fluorescence signals of a GFP fusion protein, immuno-fluorescence, and immunogold electron microscopy showed that CmstAPX was localized in the stroma of chloroplasts. Compared with wild-type plants and AtstAPX plants, CmstAPX plants were more tolerant to oxidative stress induced by methylviologen (MV, 0.4 muM) and high-temperature stress (33 degrees C). CmstAPX plants retained the highest chlorophyll content when treated with MV and high temperature, and their stroma and chloroplasts remained intact in their chloroplasts, whereas they disintegrated in wild-type plants. Our results suggest that the increased activity of APX in the chloroplasts of CmstAPX plants increased thermotolerance by increasing ROS-scavenging capacity at high temperatures.

Plant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate-rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF-MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density-gradient ultracentrifugation. The vacuole-containing fraction was subjected to SDS-PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non-lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o-methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin-tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features.

Mitochondria are not produced de novo but are maintained by division. Mitochondrial division is a coordinated process of positioning and constriction of the division site and fission of double membranes, in which dynamin-related protein is believed to mediate outer membrane fission. Part of the mitochondrial division machinery was purified from M phase-arrested Cyanidioschyzon merolae cells through biochemical fractionation. The dynamin-related protein Dnm1 was one of the two major proteins in the purified fraction and was accompanied by a newly identified protein CMR185C, named Mda1. Mda1 contained a predictable coiled-coil region and WD40 repeats, similarly to Mdv1 and Caf4 in yeasts. Immunofluorescence and immunoelectron microscopy showed that Mda1 localizes as a medial belt or ring on the mitochondrial outer surface throughout the division. The ring formation of Mda1 followed the plane of the ring of FtsZ, a protein that resides in the matrix. Dnm1 consistently colocalized with Mda1 only in the late stages of division. Mda1 protein was expressed through S to M phases and was phosphorylated specifically in M phase when Mda1 transformed from belt into foci and became colocalizing with Dnm1. Dephosphorylation of Mda1 in vitro increased its sedimentation coefficient, suggesting conformational changes of the macromolecule. Disassembly of the purified mitochondrial division machinery was performed by adding GTP to independently release Dnm1, suggesting that Mda1 forms a stable homo-oligomer by itself as a core structure of the mitochondrial division machinery.

Background All previously reported eukaryotic nuclear genome sequences have been incomplete, especially in highly repeated units and chromosomal ends. Because repetitive DNA is important for many aspects of biology, complete chromosomal structures are fundamental for understanding eukaryotic cells. Our earlier, nearly complete genome sequence of the hot-spring red alga Cyanidioschyzon merolae revealed several unique features, including just three ribosomal DNA copies, very few introns, and a small total number of genes. However, because the exact structures of certain functionally important repeated elements remained ambiguous, that sequence was not complete. Obviously, those ambiguities needed to be resolved before the unique features of the C. merolae genome could be summarized, and the ambiguities could only be resolved by completing the sequence. Therefore, we aimed to complete all previous gaps and sequence all remaining chromosomal ends, and now report the first nuclear-genome sequence for any eukaryote that is 100% complete. Results Our present complete sequence consists of 16546747 nucleotides covering 100% of the 20 linear chromosomes from telomere to telomere, representing the simple and unique chromosomal structures of the eukaryotic cell. We have unambiguously established that the C. merolae genome contains the smallest known histone-gene cluster, a unique telomeric repeat for all chromosomal ends, and an extremely low number of transposons. Conclusion By virtue of these attributes and others that we had discovered previously, C. merolae appears to have the simplest nuclear genome of the non-symbiotic eukaryotes. These unusually simple genomic features in the 100% complete genome sequence of C. merolae are extremely useful for further studies of eukaryotic cells.

Mitochondrial and chloroplast division controls the number and morphology of organelles, but how cells regulate organelle division remains to be clarified. Here, we show that each step of mitochondrial and chloroplast division is closely associated with the cell cycle in Cyanidioschyzon merolae. Electron microscopy revealed direct associations between the spindle pole bodies and mitochondria, suggesting that mitochondrial distribution is physically coupled with mitosis. Interconnected organelles were fractionated under microtubule-stabilizing condition. Immunoblotting analysis revealed that the protein levels required for organelle division increased before microtubule changes upon cell division, indicating that regulation of protein expression for organelle division is distinct from that of cytokinesis. At the mitochondrial division site, dynamin stuck to one of the divided mitochondria and was spatially associated with the tip of a microtubule stretching from the other one. Inhibition of microtubule organization, proteasome activity or DNA synthesis, respectively, induced arrested cells with divided but shrunk mitochondria, with divided and segregated mitochondria, or with incomplete mitochondrial division restrained at the final severance, and repetitive chloroplast division. The results indicated that mitochondrial morphology and segregation but not division depend on microtubules and implied that the division processes of the two organelles are regulated at distinct checkpoints.