The local distribution of both the vacuolar-type proton ATPase (V-ATPase) and the vacuolar-type proton pyrophosphatase (V-PPase), the main vacuolar proton pumps, was investigated in intact vacuoles isolated from Arabidopsis suspension-cultured cells. Fluorescent immunostaining showed that V-PPase was distributed evenly on the vacuolar membrane (VM), but V-ATPase localized to specific regions of the VM. We hypothesize that there may be membrane microdomains on the VM. To confirm this hypothesis, we prepared detergent-resistant membranes (DRMs) from the VM in accordance with well established conventional methods. Analyses of fatty acid composition suggested that DRMs had more saturated fatty acids compared with the whole VM in phosphatidylcholine and phosphatidylethanolamine. In the proteomic analyses of both DRMs and detergent-soluble mebranes (DSMs), we confirmed the different local distributions of V-ATPase and V-PPase. The observations of DRMs with an electron microscope supported the existence of different areas on the VM. Moreover, it was observed using total internal reflection fluorescent microscopy (TIRFM) that proton pumps were frequently immobilized at specific sites on the VM. In the proteomic analyses, we also found that many other vacuolar membrane proteins are distributed differently in DRMs and DSMs. Based on the results of this study, we discuss the possibility that VM microdomains might contribute to vacuolar dynamics.
Most ciliates use a deviant genetic code. Eukaryotic release factor (eRF1) appears to play an important role in the process of reassignment of stop codons. Although the precise site on eRF1 for recognition of stop codons remains obscure, studies have suggested that the tip region NIKS and its adjacent YxCxxxF motifs in domain 1 are important for stop codon recognition. Litostomatea is a class of ciliate that appears to use the standard genetic code. We used Dileptus (Litostomatea) eRF1 in this study to identify key residues located in or near the YxCxxxF motif. We predicted sites involving stop codon recognition by computational calculation of RNA-protein interaction propensity. We introduced mutations at the predicted sites of Dileptus eRF1 and examined the activity of the mutated Dileptus eRF1 using in vivo assay systems. The results show that the single mutation R128I (Dileptus eRF1 numbering) in the YxCxxxF motif converted the omnipotent recognition of Dileptus eRF1 to Euplotes-type dualpotent eRF1. Our results indicate that R128 is one of the key residues preserving the ability to recognize all three stop codons, especially UGA, in Dileptus. We discuss a possible advantage that ciliates from the Litostomatea class may gain from using the standard genetic code.
Euglenophytes are a group of photosynthetic flagellates possessing a plastid derived from a green algal endosymbiont, which was incorporated into an ancestral host cell via secondary endosymbiosis. However, the impact of endosymbiosis on the euglenophyte nuclear genome is not fully understood due to its complex nature as a hybrid of a non-photosynthetic host cell and a secondary endosymbiont.
In contrast to most ciliates, meiosis and successive nuclear changes during conjugation occur only in heterotypic pairs in Blepharisma. It has been suggested that homotypic pairs are ready for conjugation, but lack a trigger to initiate the nuclear changes, and the conjugation process is arrested before the onset of meiosis. To explore the possible nature of the trigger, we previously identified the genes BjCdk1 (homologous to cdk1/cdc2), Bj4HPPD (4-hydroxy-phenylpyruvate dioxygenase) and BjCks (cyclin dependent kinase regulatory subunit) whose expression is up-regulated in gamone1-treated type II cells. In this study, we investigated the molecular structures of these three genes, and compared their expression patterns in homotypic and heterotypic pairs, finding remarkable differences. BjCdk1, Bj4HPPD and BjCks were expressed specifically in gamone1-treated type II cells, but not in gamone2-treated type I cells. In heterotypic pairs, the expression of these genes stayed at the same level or gradually decreased throughout the entire process of conjugation, but it rapidly decreased and ceased after 10hours in homotypic pairs. These results indicate that some genes are expressed in a mating-type specific manner. Alternative gene expression in mating type I and type II cells and merging of individual factors in a heterotypic pair may induce nuclear changes including meiosis.
Preconjugant interactions between complementary mating-type cells in ciliates occur before sexual reproduction. The interactions include retardation of swimming behaviour, courtship dancing, chemoattraction, nuclear activation, cell division, or cell agglutination, depending on ciliate species. In Blepharisma japonicum, chemoattraction of mating-type I by mating-type II has been reported previously. It has been shown that chemoattraction here is caused by a conjugation-inducing substance called gamone 2 secreted by mating-type II cells. In this study, we show that mating-type II cells accumulate near the site where gamone 1 secreted by mating-type I cells is present at a high concentration. We also show that the behaviour of individual cells changes when exposed to the complementary mating-type gamone; cells begin to rotate and swim slowly, thus shortening their minimum path length (final displacement of a cell from its origin). These results suggest that gamones 1 and 2 induce behavioural changes in type II and I cells, respectively, and that gamone-stimulated cells may accumulate at the site with the highest activity of the complementary gamone, after repetition of swimming changes in the gradient of gamone concentration. This reciprocal induction of the changes in behaviour may increase the probability of sexual encounters for conjugation.
Tripartite tubular hairs (mastigonemes) on the anterior flagellum of protists in the stramenopile taxon are responsible for reversing the thrust of flagellar beat and for cell motility. Immunoprecipitation experiments using antibodies directed towards mastigonemes on the flagella of zoospores ofPhytophthora nicotianaehave facilitated the cloning of a gene encoding a mastigoneme shaft protein in this Oomycete. Expression of the gene, designatedPnMas2, is up-regulated during asexual sporulation, a period during which many zoospore components are synthesized. Analysis of the sequence of the PnMas2 protein has revealed that, like other stramenopile mastigoneme proteins, PnMas2 has an N-terminal secretion signal and contains four cysteine-rich epidermal growth factor (EGF)-like domains. Evidence from non-denaturing gels indicates that PnMas2 forms large oligomeric complexes, most likely through disulphide bridging. Bioinformatic analysis has revealed thatPhytophthoraspecies typically contain three or four putative mastigoneme proteins containing the four EGF-like domains. These proteins are similar in sequence to mastigoneme proteins in other stramenopile protists including the algaeOchromonas danica,Aureococcus anophagefferensandScytosiphon lomentariaand the diatomsThalassiosira pseudonana and T. weissflogii.
Recent multigene phylogenetic analyses have contributed much to our understanding of eukaryotic phylogeny. However, the phylogenetic positions of various lineages within the eukaryotes have remained unresolved or in conflict between different phylogenetic studies. These phylogenetic ambiguities might have resulted from mixtures or integration from various factors including limited taxon sampling, missing data in the alignment, saturations of rapidly evolving genes, mixed analyses of short- and long-branched operational taxonomic units (OTUs), intracellular endoparasite and ciliate OTUs with unusual substitution etc. In order to evaluate the effects from intracellular endoparasite and ciliate OTUs co-analyzed on the eukaryotic phylogeny and simplify the results, we here used two different sets of data matrices of multiple slowly evolving genes with small amounts of missing data and examined the phylogenetic position of the secondary photosynthetic chromalveolates Haptophyta, one of the most abundant groups of oceanic phytoplankton and significant primary producers. In both sets, a robust sister relationship between Haptophyta and SAR (stramenopiles, alveolates, rhizarians, or SA [stramenopiles and alveolates]) was resolved when intracellular endoparasite/ciliate OTUs were excluded, but not in their presence. Based on comparisons of character optimizations on a fixed tree (with a clade composed of haptophytes and SAR or SA), disruption of the monophyly between haptophytes and SAR (or SA) in the presence of intracellular endoparasite/ciliate OTUs can be considered to be a result of multiple evolutionary reversals of character positions that supported the synapomorphy of the haptophyte and SAR (or SA) clade in the absence of intracellular endoparasite/ciliate OTUs.
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