Glycosyltransferase MDR1 assembles a dividing ring for mitochondrial proliferation comprising polyglucan nanofilaments.

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.

Defining the dynamin-based ring organizing center on the peroxisome-dividing machinery isolated from Cyanidioschyzon merolae.

Organelle division is executed through contraction of a ring-shaped supramolecular dividing machinery. A core component of the machinery is the dynamin-based ring conserved during the division of mitochondrion, plastid and peroxisome. Here, using isolated peroxisome-dividing (POD) machinery from a unicellular red algae, Cyanidioschyzon merolae, we identified a dynamin-based ring organizing center (DOC) that acts as an initiation point for formation of the dynamin-based ring. C. merolae contains a single peroxisome, the division of which can be highly synchronized by light-dark stimulation; thus, intact POD machinery can be isolated in bulk. Dynamin-based ring homeostasis is maintained by the turnover of the GTP-bound form of the dynamin-related protein Dnm1 between the cytosol and division machinery via the DOC. A single DOC is formed on the POD machinery with a diameter of 500-700 nm, and the dynamin-based ring is unidirectionally elongated from the DOC in a manner that is dependent on GTP concentration. During the later step of membrane fission, the second DOC is formed and constructs the double dynamin-based ring to make the machinery thicker. These findings provide new insights to define fundamental mechanisms underlying the dynamin-based membrane fission in eukaryotic cells.

The sole LSm complex in Cyanidioschyzon merolae associates with pre-mRNA splicing and mRNA degradation factors.

Proteins of the Sm and Sm-like (LSm) families, referred to collectively as (L)Sm proteins, are found in all three domains of life and are known to promote a variety of RNA processes such as base-pair formation, unwinding, RNA degradation, and RNA stabilization. In eukaryotes, (L)Sm proteins have been studied, inter alia, for their role in pre-mRNA splicing. In many organisms, the LSm proteins form two distinct complexes, one consisting of LSm1-7 that is involved in mRNA degradation in the cytoplasm, and the other consisting of LSm2-8 that binds spliceosomal U6 snRNA in the nucleus. We recently characterized the splicing proteins from the red alga Cyanidioschyzon merolae and found that it has only seven LSm proteins. The identities of CmLSm2-CmLSm7 were unambiguous, but the seventh protein was similar to LSm1 and LSm8. Here, we use in vitro binding measurements, microscopy, and affinity purification-mass spectrometry to demonstrate a canonical splicing function for the C. merolae LSm complex and experimentally validate our bioinformatic predictions of a reduced spliceosome in this organism. Copurification of Pat1 and its associated mRNA degradation proteins with the LSm proteins, along with evidence of a cytoplasmic fraction of CmLSm complexes, argues that this complex is involved in both splicing and cytoplasmic mRNA degradation. Intriguingly, the Pat1 complex also copurifies with all four snRNAs, suggesting the possibility of a spliceosome-associated pre-mRNA degradation complex in the nucleus.

Dynamics of the nucleoside diphosphate kinase protein DYNAMO2 correlates with the changes in the global GTP level during the cell cycle of Cyanidioschyzon merolae.

GTP is an essential source of energy that supports a large array of cellular mechanochemical structures ranging from protein synthesis machinery to cytoskeletal apparatus for maintaining the cell cycle. However, GTP regulation during the cell cycle has been difficult to investigate because of heterogenous levels of GTP in asynchronous cell cycles and genetic redundancy of the GTP-generating enzymes. Here, in the unicellular red algae Cyanidioschyzon merolae, we demonstrated that the ATP-GTP-converting enzyme DYNAMO2 is an essential regulator of global GTP levels during the cell cycle. The cell cycle of C. merolae can be highly synchronized by light/dark stimulations to examine GTP levels at desired time points. Importantly, the genome of C. merolae encodes only two isoforms of the ATP-GTP-converting enzyme, namely DYNAMO1 and DYNAMO2. DYNAMO1 regulates organelle divisions, whereas DYNAMO2 is entirely localized in the cytoplasm. DYNAMO2 protein levels increase during the S-M phases, and changes in GTP levels are correlated with these DYNAMO2 protein levels. These results indicate that DYNAMO2 is a potential regulator of global GTP levels during the cell cycle.

Single-membrane-bounded peroxisome division revealed by isolation of dynamin-based machinery.

Peroxisomes (microbodies) are ubiquitous single-membrane-bounded organelles and fulfill essential roles in the cellular metabolism. They are found in virtually all eukaryotic cells and basically multiply by division. However, the mechanochemical machinery involved in peroxisome division remains elusive. Here, we first identified the peroxisome-dividing (POD) machinery. We isolated the POD machinery from Cyanidioschyzon merolae, a unicellular red alga containing a single peroxisome. Peroxisomal division in C. merolae can be highly synchronized by light/dark cycles and the microtubule-disrupting agent oryzalin. By proteomic analysis based on the complete genome sequence of C. merolae, we identified a dynamin-related protein 3 (DRP3) ortholog, CmDnm1 (Dnm1), that predominantly accumulated with catalase in the dividing-peroxisome fraction. Immunofluorescence microscopy demonstrated that Dnm1 formed a ring at the division site of the peroxisome. The outlines of the isolated dynamin rings were dimly observed by phase-contrast microscopy and clearly stained for Dnm1. Electron microscopy revealed that the POD machinery was formed at the cytoplasmic side of the equator. Immunoelectron microscopy showed that the POD machinery consisted of an outer dynamin-based ring and an inner filamentous ring. Down-regulation of Dnm1 impaired peroxisomal division. Surprisingly, the same Dnm1 serially controlled peroxisomal division after mitochondrial division. Because genetic deficiencies of Dnm1 orthologs in multiperoxisomal organisms inhibited both mitochondrial and peroxisomal proliferation, it is thought that peroxisomal division by contraction of a dynamin-based machinery is universal among eukaryotes. These findings are useful for understanding the fundamental systems in eukaryotic cells.

The kinesin-like protein TOP promotes Aurora localisation and induces mitochondrial, chloroplast and nuclear division.

The cell cycle usually refers to the mitotic cycle, but the cell-division cycle in the plant kingdom consists of not only nuclear but also mitochondrial and chloroplast division cycle. However, an integrated control system that initiates division of the three organelles has not been found. We report that a novel C-terminal kinesin-like protein, three-organelle division-inducing protein (TOP), controls nuclear, mitochondrial and chloroplast divisions in the red alga Cyanidioschyzon merolae. A proteomics study revealed that TOP is a member of a complex of mitochondrial-dividing (MD) and plastid-dividing (PD) machineries (MD/PD machinery complex) just prior to constriction. After TOP localizes at the MD/PD machinery complex, mitochondrial and chloroplast divisions occur and the components of the MD/PD machinery complexes are phosphorylated. Furthermore, we found that TOP downregulation impaired both mitochondrial and chloroplast divisions. MD/PD machinery complexes were formed normally at each division site but they were neither phosphorylated nor constricted in these cells. Immunofluorescence signals of Aurora kinase (AUR) were localized around the MD machinery before constriction, whereas AUR was dispersed in the cytosol by TOP downregulation, suggesting that AUR is required for the constriction. Taken together our results suggest that TOP induces phosphorylation of MD/PD machinery components to accomplish mitochondrial and chloroplast divisions prior to nuclear division, by relocalization of AUR. In addition, given the presence of TOP homologs throughout the eukaryotes, and the involvement of TOP in mitochondrial and chloroplast division may illuminate the original function of C-terminal kinesin-like proteins.

Complete mitochondrial and chloroplast DNA sequences of the freshwater green microalga Medakamo hakoo.

We report the complete organellar genome sequences of an ultrasmall green alga, Medakamo hakoo strain M-hakoo 311, which has the smallest known nuclear genome in freshwater green algae. Medakamo hakoo has 90.8-kb chloroplast and 36.5-kb mitochondrial genomes containing 80 and 33 putative protein-coding genes, respectively. The mitochondrial genome is the smallest in the Trebouxiophyceae algae studied so far. The GC content of the nuclear genome is 73%, but those of chloroplast and mitochondrial genomes are 41% and 35%, respectively. Codon usages in the organellar genomes have a different tendency from that in the nuclear genome. The organellar genomes have unique characteristics, such as the biased encoding of mitochondrial genes on a single strand and the absence of operon structures in chloroplast ribosomal genes. Medakamo hakoo will be helpful for understanding the evolution of the organellar genome and the regulation of gene expression in chloroplasts and mitochondria.

The coiled-coil protein VIG1 is essential for tethering vacuoles to mitochondria during vacuole inheritance of Cyanidioschyzon merolae.

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.

GENERATIVE CELL SPECIFIC 1 is essential for angiosperm fertilization.

The double fertilization process in angiosperms is based on the delivery of a pair of sperm cells by the pollen tube (the male gametophyte), which elongates towards an embryo sac (the female gametophyte) enclosing an egg and a central cell. Several studies have described the mechanisms of gametophyte interaction, and also the fertilization process - from pollination to pollen tube acceptance. However, the mechanisms of gamete interaction are not fully understood. Cytological studies have shown that male gametes possess distinct cell-surface structures and genes specific to male gametes have been detected in cDNA libraries. Thus, studies of isolated gametes may offer clues to understanding the sperm-egg interaction. In this study, we identified a novel protein, designated GCS1 (GENERATIVE CELL SPECIFIC 1), using generative cells isolated from Lilium longiflorum pollen. GCS1 possesses a carboxy-terminal transmembrane domain, and homologues are present in various species, including non-angiosperms. Immunological assays indicate that GCS1 is accumulated during late gametogenesis and is localized on the plasma membrane of generative cells. In addition, Arabidopsis thaliana GCS1 mutant gametes fail to fuse, resulting in male sterility and suggesting that GCS1 is a critical fertilization factor in angiosperms.

Genomic analysis of an ultrasmall freshwater green alga, Medakamo hakoo.

Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmall algae remain genetically uncharacterized. Here, we present the nuclear genome sequence of an ultrasmall green alga of freshwater habitats, Medakamo hakoo. Evolutionary analyses suggest that this species belongs to a new genus within the class Trebouxiophyceae. Sequencing analyses revealed that its genome, comprising 15.8 Mbp and 7629 genes, is among the smallest known genomes in the Viridiplantae. Its genome has relatively few genes associated with genetic information processing, basal transcription factors, and RNA transport. Comparative analyses revealed that 1263 orthogroups were shared among 15 ultrasmall algae from distinct phylogenetic lineages. The shared gene sets will enable identification of genes essential for algal metabolism and cellular functions.

The soybean F3'H protein is localized to the tonoplast in the seed coat hilum.

We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3'-hydroxylase (F3'H) gene (sf3'h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3'h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3'h1 was not detected in To7G. A truncated sf3'h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3'H was detected using microsomal fractions from yeast transformed with sf3'h1 from To7B, but not from To7G. These results indicate that sf3'h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3'h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3'h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3'H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.

Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells.

Eukaryotic cells arose from an ancient endosymbiotic association of prokaryotes, with plant cells harboring 3 genomes as the remnants of such evolution. In plant cells, plastid and mitochondrial DNA replication [organelle DNA replication (ODR)] occurs in advance of the subsequent cell cycles composed of nuclear DNA replication (NDR) and cell division. However, the mechanism by which replication of these genomes with different origins is coordinated is largely unknown. Here, we show that NDR is regulated by a tetrapyrrole signal in plant cells, which has been suggested as an organelle-to-nucleus retrograde signal. In synchronized cultures of the primitive red alga Cyanidioschyzon merolae, specific inhibition of A-type cyclin-dependent kinase (CDKA) prevented NDR but not ODR after onset of the cell cycle. In contrast, inhibition of ODR by nalidixic acid also resulted in inhibition of NDR, indicating a strict dependence of NDR on ODR. The requirement of ODR for NDR was bypassed by addition of the tetrapyrrole intermediates protoporphyrin IX (ProtoIX) or Mg-ProtoIX, both of which activated CDKA without inducing ODR. This scheme was also observed in cultured tobacco cells (BY-2), where inhibition of ODR by nalidixic acid prevented CDKA activation and NDR, and these inhibitions were circumvented by Mg-ProtoIX without inducing ODR. We thus show that tetrapyrrole-mediated organelle-nucleus replicational coupling is an evolutionary conserved process among plant cells.

Existence of giant mitochondria-containing sheet structures lacking cristae and matrix in the etiolated cotyledon of Arabidopsis thaliana.

Mitochondria are essential organelles involved in the production and supply of energy in eukaryotic cells. Recently, the use of serial section scanning electron microscopy (S3EM) has allowed accurate three-dimensional (3D) reconstructed images of even complex organelle structures. Using this method, ultrathin sections of etiolated cotyledons were observed 4 days after germination of Arabidopsis thaliana in the dark, and giant mitochondria were found. To exclude the possibility of chemical fixation artifacts, this study confirmed the presence of giant mitochondria in high-pressure frozen samples. The 3D reconstructed giant mitochondria had a complex structure that included not only the elongated region but also the flattened shape of a disk. It contained the characteristic sheet structure, and the sheet lacked cristae and matrix but consisted of outer and inner membranes. Whether this phenomenon could be observed in living cells was investigated using the transformant with mitochondrial matrix expressing green fluorescent protein. Small globular mitochondria observed in light-treated samples were also represented in etiolated cotyledons. Although no giant mitochondria were observed in light-treated samples, they were found in the dark 3 days after germination and rapidly increased in number on the fourth day. Therefore, giant mitochondria were observed only in dark samples. These findings were supported by electron microscopy results.

The cell cycle, including the mitotic cycle and organelle division cycles, as revealed by cytological observations.

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.

Identification of novel proteins in isolated polyphosphate vacuoles in the primitive red alga Cyanidioschyzon merolae.

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.

Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D.

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.

Evolutionary significance of the ring-like plastid nucleus in the primitive red alga Cyanidioschyzon merolae as revealed by drying.

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.

Efficient open cultivation of cyanidialean red algae in acidified seawater.

Microalgae possess high potential for producing pigments, antioxidants, and lipophilic compounds for industrial applications. However, their open pond cultures are often contaminated by other undesirable organisms, including their predators. In addition, the cost of using freshwater is relatively high, which limits the location and scale of cultivation compared with using seawater. It was previously shown that Cyanidium caldarium and Galdieria sulphuraria, but not Cyanidioschyzon merolae grew in media containing NaCl at a concentration equivalent to seawater. We found that the preculture of C. merolae in the presence of a moderate NaCl concentration enabled the cells to grow in the seawater-based medium. The cultivation of cyanidialean red algae in the seawater-based medium did not require additional pH buffering chemicals. In addition, the combination of seawater and acidic conditions reduced the risk of contamination by other organisms in the nonsterile open culture of C. merolae more efficiently than the acidic condition alone.

ESCRT Machinery Mediates Cytokinetic Abscission in the Unicellular Red Alga Cyanidioschyzon merolae.

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.

A cryptic algal group unveiled: a plastid biosynthesis pathway in the oyster parasite Perkinsus marinus.

Plastids are widespread in plant and algal lineages. They are also exploited by some nonphotosynthetic protists, including malarial parasites, to support their diverse modes of life. However, cryptic plastids may exist in other nonphotosynthetic protists, which could be important in studies on the diversity and evolution of plastids. The parasite Perkinsus marinus, which causes mass mortality in oyster farms, is a nonphotosynthetic protist that is phylogenetically related to plastid-bearing dinoflagellates and apicomplexans. In this study, we searched for P. marinus methylerythritol phosphate (MEP) pathway genes, responsible for de novo isoprenoid synthesis in plastids, and determined the full-length gene sequences for 6 of 7 of these genes. Phylogenetic analyses revealed that each P. marinus gene clusters with orthologs from plastid-bearing eukaryotes, which have MEP pathway genes with essentially the same mosaic pattern of evolutionary origin. A new analytical method called sliding-window iteration of TargetP was developed to examine the distribution of targeting preferences. This analysis revealed that the sequenced genes encode bipartite targeting peptides that are characteristic of proteins targeted to secondary plastids originating from endosymbiosis of eukaryotic algae. These results support our idea that Perkinsus is a cryptic algal group containing nonphotosynthetic secondary plastids. In fact, immunofluorescent microscopy indicated that 1 of the MEP pathway enzymes, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, was localized to small compartments near mitochondrion, which are possibly plastids. This tiny organelle seems to contain very low quantities of DNA or may even lack DNA entirely. The MEP pathway genes are a useful tool for investigating plastid evolution in both of the photosynthetic and nonphotosynthetic eukaryotes and led us to propose the hypothesis that ancestral "chromalveolates" harbored plastids before a secondary endosymbiotic event.