Proteins In Tetrahymena Research Articles

Tethering CST to the telomerase catalytic core—the p50 linchpin

Emerging models of telomere and associated proteins in Tetrahymena have broadened our understanding oftelomeric processes. In this issue of Structure, Ma etal. pinpoint a region in p50 that associates with the CST complex. The interaction mediated by p50 is crucial for the optimal positioning of TERT to maintain homeostasis at the chromosome ends.

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo-EM.

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo-electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse-grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.

Spatial constraints on chromosomes are instrumental to meiotic pairing.

ABSTRACTIn most eukaryotes, the meiotic chromosomal bouquet (comprising clustered chromosome ends) provides an ordered chromosome arrangement that facilitates pairing and recombination between homologous chromosomes. In the protist Tetrahymena thermophila, the meiotic prophase nucleus stretches enormously, and chromosomes assume a bouquet-like arrangement in which telomeres and centromeres are attached to opposite poles of the nucleus. We have identified and characterized three meiosis-specific genes [meiotic nuclear elongation 1-3 (MELG1-3)] that control nuclear elongation, and centromere and telomere clustering. The Melg proteins interact with cytoskeletal and telomere-associated proteins, and probably repurpose them for reorganizing the meiotic prophase nucleus. A lack of sequence similarity between the Tetrahymena proteins responsible for telomere clustering and bouquet proteins of other organisms suggests that the Tetrahymena bouquet is analogous, rather than homologous, to the conserved eukaryotic bouquet. We also report that centromere clustering is more important than telomere clustering for homologous pairing. Therefore, we speculate that centromere clustering may have been the primordial mechanism for chromosome pairing in early eukaryotes.

Grl1 Protein is a Candidate K Antigen in Tetrahymena thermophila

In Tetrahymena, K antigens associate only with mature basal bodies and are expected to play important roles in the morphogenesis and function of the membrane skeleton around basal bodies, but these proteins have not been identified and their functions are unknown. Commercially available anti-human Rho GDP-dissociation inhibitor α (RhoGDIα) antibody (sc-33201) was accidentally found to show very similar immunofluorescence staining patterns to those of anti-K antigen antibodies, such as 424A8 and 10D12 mouse monoclonal antibodies, in Tetrahymena. A 40kDa protein recognized by this antibody was partially purified and identified as granule lattice protein 1 (Grl1p) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. In immunoblotting experiments this antibody was suggested to recognize endogenous Grl1p. The three-dimensional structure of proGrl1p protein predicted by I-TASSER was similar to a spectrin family protein. Grl1 may be a K antigen and a spectrin-like protein in Tetrahymena.

Tetrahymena dynamin-related protein 6 self-assembles independent of membrane association

Self-assembly on target membranes is one of the important properties of all dynamin family proteins. Drp6, a dynaminrelated protein in Tetrahymena, controls nuclear remodelling and undergoes cycles of assembly/disassembly on the nuclear envelope. To elucidate the mechanism of Drp6 function, we have characterized its biochemical and biophysical properties using size exclusion chromatography, chemical cross-linking and electron microscopy. The results demonstrate that Drp6 readily forms high-molecular-weight self-assembled structures as determined by size exclusion chromatography and chemical cross-linking. Negative stain electron microscopy revealed that Drp6 assembles into rings and spirals at physiological ionic strength. We have also shown that the recombinant Drp6 expressed in bacteria is catalytically active and its GTPase activity is not enhanced by low salt. These results suggest that, in contrast to dynamins but similar to MxA, Drp6 self-assembles in the absence of membrane templates, and its GTPase activity is not affected by ionic strength of the buffer. We discuss the self-assembly structure of Drp6 and explain the basis for lack of membrane-stimulated GTPase activity.

Sfr1, a Tetrahymena thermophila Sfi1 Repeat Protein, Modulates the Production of Cortical Row Basal Bodies.

Basal bodies are essential microtubule-based structures that template, anchor, and orient cilia at the cell surface. Cilia act primarily in the generation of directional fluid flow and sensory reception, both of which are utilized for a broad spectrum of cellular processes. Although basal bodies contribute to vital cell functions, the molecular contributors of their assembly and maintenance are poorly understood. Previous studies of the ciliate Tetrahymena thermophila revealed important roles for two centrin family members in basal body assembly, separation of new basal bodies, and stability. Here, we characterize the basal body function of a centrin-binding protein, Sfr1, in Tetrahymena. Sfr1 is part of a large family of 13 proteins in Tetrahymena that contain Sfi1 repeats (SFRs), a motif originally identified in Saccharomyces cerevisiae Sfi1 that binds centrin. Sfr1 is the only SFR protein in Tetrahymena that localizes to all cortical row and oral apparatus basal bodies. In addition, Sfr1 resides predominantly at the microtubule scaffold from the proximal cartwheel to the distal transition zone. Complete genomic knockout of SFR1 (sfr1Δ) causes a significant increase in both cortical row basal body density and the number of cortical rows, contributing to an overall overproduction of basal bodies. Reintroduction of Sfr1 into sfr1Δ mutant cells leads to a marked reduction of cortical row basal body density and the total number of cortical row basal bodies. Therefore, Sfr1 directly modulates cortical row basal body production. This study reveals an inhibitory role for Sfr1, and potentially centrins, in Tetrahymena basal body production. IMPORTANCE Basal bodies and centrioles are structurally similar and, when rendered dysfunctional as a result of improper assembly or maintenance, are associated with human diseases. Centrins are conserved and abundant components of both structures whose basal body and centriolar functions remain incompletely understood. Despite the extensive study of centrins in Tetrahymena thermophila, little is known about how centrin-binding proteins contribute to centrin's roles in basal body assembly, stability, and orientation. The sole previous study of the large centrin-binding protein family in Tetrahymena revealed a role for Sfr13 in the stabilization and separation of basal bodies. In this study, we found that Sfr1 localizes to all Tetrahymena basal bodies and complete genetic deletion of SFR1 leads to overproduction of basal bodies. The uncovered inhibitory role of Sfr1 in basal body production suggests that centrin-binding proteins, as well as centrins, may influence basal body number both positively and negatively.

Identification of biotin carboxyl carrier protein in Tetrahymena and its application in in vitro motility systems of outer arm dynein

Axonemal dynein plays a central role in ciliary beating. Recently, a functional expression system of axonemal dynein was established in the ciliated protozoan Tetrahymena. This study identifies biotin carboxyl carrier protein (BCCP) in Tetrahymena and demonstrates its application in in vitro motility systems of outer arm dynein.

Sfr13 is a member of a large family of asymmetrically 1 localized Sfi1-repeat proteins and is important for basal body separation and stability inTetrahymena thermophila

Directed fluid flow, which is achieved by the coordinated beating of motile cilia, is required for processes as diverse as cellular swimming, developmental patterning and mucus clearance. Cilia are nucleated, anchored and aligned at the plasma membrane by basal bodies, which are cylindrical microtubule-based structures with ninefold radial symmetry. In the unicellular ciliate Tetrahymena thermophila, two centrin family members associated with the basal body are important for both basal body organization and stabilization. We have identified a family of 13 proteins in Tetrahymena that contain centrin-binding repeats related to those identified in the Saccharomyces cerevisiae Sfi1 protein. We have named these proteins Sfr1-Sfr13 (for Sfi1-repeat). Nine of the Sfr proteins localize in unique polarized patterns surrounding the basal body, suggesting non-identical roles in basal body organization and association with basal body accessory structures. Furthermore, the Sfr proteins are found in distinct basal body populations in Tetrahymena cells, indicating that they are responsive to particular developmental programs. A complete genetic deletion of one of the family members, Sfr13, causes unstable basal bodies and defects in daughter basal body separation from the mother, phenotypes also observed with centrin disruption. It is likely that the other Sfr family members are involved in distinct centrin functions, providing specificity to the tasks that centrins perform at basal bodies.

Characterization of affinity tag features of recombinant Tetrahymena thermophila glutathione-S-transferase zeta for Tetrahymena protein expression vectors

Glutathione S-transferase (GST) is the one of most widely used affinity tags in biotechnology applications. The present study named a GST gene as the TtGSTz1 gene, characterized with the conserved glutathione binding motif (SSTSWRVRIAL) under a GST zeta subfamily from Tetrahymena thermophila. Phylogenetic analysis of the TtGstz1p protein sequence, with its orthologs from different GST classes, showed that it is a member of the unicellular GSTz monophyletic clade, which is positioned close to the bacterial GSTz clade. Seven codons of the TtGSTz1 gene were first engineered by introducing silent mutations (TAA > CAA or TAG > CAG) to the code for glutamine instead of stop signals in E. coli. The recombinant TtGSTz1 gene, with its 6XHis tag, was expressed using the pET16b expression plasmid and E. coli BL21(DE3). SDS-PAGE analysis of the 6XHis-TtGstz1p protein confirmed its expression and purification by nickel agarose and glutathione-sepharose 4B. Additionally, both anti-His and anti-GST antibodies recognized the purified 6XHis-TtGSTz in the western blot analysis. In conclusion, the results presented here suggest that the 6XHis-TtGstz1p fusion protein could be used as a dual tag in Tetrahymena expression vectors with a sequential 2-step protein purification procedure.

Multifunctional acyltransferases from Tetrahymena thermophila.

Multifunctional acyltransferases are able to catalyze the esterification of various acyl-acceptors with activated fatty acids. Here we describe the identification of four proteins from Tetrahymena thermophila that share certain properties with mammalian acyltransferases regarding their predicted transmembrane structure, their molecular mass and the typical acyltransferase motif. Expression of the Tetrahymena sequences results in production of triacylglycerols and wax esters in recombinant yeast when appropriate substrates are provided. The in vitro characterization shows, that these enzymes are capable of esterifying different acyl-acceptors including fatty alcohols, diols, diacylglycerols and isoprenols with acyl-CoA thioesters. Based on these catalytic activities and the sequence similarities of the Tetrahymena proteins with acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) family members, we conclude that we identified a new group of DGAT2-related multifunctional acyltransferases from protozoan organisms.Electronic supplementary materialThe online version of this article (doi:10.1007/s11745-011-3642-1) contains supplementary material, which is available to authorized users.

A Novel Robust Heat-inducible Promoter for Heterologous Gene Expression in Tetrahymena thermophila

An increasing amount of data has revealed the importance of inducible promoters in ciliate research and in ciliate-related industries. However, knowledge about these promoters and related genes is relatively sparse. Here we report a novel inducible promoter from a Tetrahymena cytoplasmic Hsp70 gene member, HSP70-2. The reported promoter was able to induce the endogenous gene up to ∼9000-fold after a short heat shock treatment and this remarkable feature has been retained when a relatively short region of the promoter was introduced into a reporter construct followed by transformation. During the recovery period following a short heat shock, both the mRNA and protein levels of the reporter gene were maintained high up to two hours. A constant heat shock treatment to the transformed cells led to a stabilization of the reporter mRNA up to at least six hours and the reporter protein continued to accumulate up to around three hours. The promoter strength appears to be similar to that of the cadmium-induced metallothionein gene (MTT1) promoter. Therefore, the HSP70-2 promoter represents an attractive alternative for the over-expression of proteins in Tetrahymena, and the promoter-reporter gene construct used in this study is an ideal tool to help in understanding the regulation mechanisms of heat shock genes in ciliates.

Recombinant protein expression of Tetrahymena thermophila glutathione-S-transferase zeta (6xHis-GSTz) in E. coli suggest a possible use as a GSTz or 6xHis-GSTz dual tag in Tetrahymena protein expression vectors

Recombinant protein expression of Tetrahymena thermophila glutathione-S-transferase zeta (6xHis-GSTz) in E. coli suggest a possible use as a GSTz or 6xHis-GSTz dual tag in Tetrahymena protein expression vectors

Identification of a novel actin-related protein inTetrahymena cilia

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In eukaryotes, its gene belongs to a highly conserved gene family, while the protein is also a member of an actin superfamily comprising various kinds of actin-related proteins (Arps). A ciliate, Tetrahymena, has a unique conventional actin. Data from the TIGR Tetrahymena genome project and our own research suggest the existence of 12 actin-like sequences: four conventional actins, two of Arp4, one each of Arp1, Arp2, Arp3, Arp5, and Arp6, and a novel actin-related protein, tArp. We cloned the entire cDNA sequences of Tetrahymena Arp2 (tArp2), Tetrahymena Arp3 (tArp3), and tArp for the work described herein. In phylogenetic analyses, tArp was not included in any Arp subfamily. Unlike other known Arps, tArp localizes in cilia, and its expression was upregulated after deciliation. To see the precise localization of tArp, cilia were fractionated and analyzed using specific antibodies. tArp was observed preferentially in the "outer-doublet" fraction, while actin was found in the "crude-dynein" fraction. In immunoelectron microscopy, most of the gold particles were found either on the outer-doublet or central-pair microtubules. These results suggest that tArp is a ciliary component and that it has a unique function in the formation and maintenance of cilia.

Fine oral filaments in Paramecium: a biochemical and immunological analysis.

In Paramecium, several kinds of the oral networks of fine filaments are defined at the ultrastructural level. Using the sodium chloride-treated oral apparatus of Paramecium as an antigen to produce monoclonal antibodies, we have begun to identify the proteins constituting these networks. Immunoblotting showed that all positive antibodies were directed against three bands (70-, 75-and 83-kD), which corresponded to quantitatively minor components of the antigen; there was no antibody specific for the quantitatively major components (58- and 62-kD). Immunolocalization with four of these antibodies directed against one or several of these three bands showed that these proteins are components of the fine filaments supporting the oral area; a decoration of the basal bodies and the outer lattice was also observed on the cortex. Immunofluorescence on interphase cells suggested that the three proteins colocalized on the left side of the oral apparatus, whereas only the 70-kD band was detected on the right side. During division, the antigens of the antibodies were detected at different stages after oral basal body assembly. The antibodies cross-reacted with the tetrins, which are oral filament-forming proteins in Tetrahymena, demonstrating that tetrin-related proteins are quantitatively minor components of the oral and the somatic cytoskeleton of Paramecium.

Cadmium metallothionein gene of Tetrahymena pyriformis.

A genomic sequence from Tetrahymena pyriformis, encoding a cadmium-induced metallothionein has been cloned. The gene encodes a transcript of 487 bases, with an intronless coding region of 324 nt, using TGA as the stop codon, TAA coding for glutamine, and the translational initiation sequence AAAATGG. Two regions of internal similarity in the coding sequence support the hypothesis that the Tetrahymena protein arose by gene duplication. The sequences of untranslated regions show some similarities with nematode MT-1 and MT-2 transcripts. Sequence of 525 bases upstream of the transcription start contains a TATA box, a CAAT box reverse complement, and many short stretches partially matching the AP-1 and ACE-1 binding sites, but no characteristic sequences found in other metallothionein promoters.

Tetrahymena Elongation Factor-1α Binds to Hsp70 Family Proteins

Abstract Translation elongation factor 1α (EF-1α) catalyzes the GTP-dependent binding of aminoacyl-tRNA to the ribosome. We previously reported that Tetrahymena EF-1α induced bundles of rabbit skeletal muscle F-actin as well as Tetrahymena F-actin (Kurasawa et al., (1996) Zool. Sci. 13: 371–375), and that Ca2+/calmodulin (CaM) regulated the F-actin-bundling activity of EF-1α without inhibition of the binding between EF-1α and F-actin (Kurasawa et al., (1996) J. Biochem. 119: 791–798). In this study, we investigated EF-1α-binding proteins in Tetrahymena using a Tetrahymena EF-1α affinity column. Tetrahymena EF-1α bound directly to 74 kDa, 77 kDa, and 78 kDa proteins, in addition to CaM. The bindings of 74 kDa, 77 kDa, and 78 kDa proteins to Tetrahymena EF-1α were Ca2+-independent and ATP-sensitive. The N-terminal amino acid sequence of the 74 kDa protein was similar to those of 70 kDa heat shock protein (hsp70) family.

Chapter 15 Immunoelectron Microscopy of Tetrahymena

Publisher Summary This chapter focuses on the immunogold labeling technique for transmission electron microscopy. Pre-embedding and post-embedding labeling protocols with a single primary antibody and a double labeling protocol with two different primary antibodies are described. The labeling protocols include a description of a morphometric analysis to quantitate colloidal gold distribution in labeling experiments. Colloidal gold-labeled antisera are widely used to map intracellular distribution of antigens. To use this technique, a primary antibody is bound to antigenic sites in situ , and a secondary antibody, coupled to colloidal gold and specific for the general class of the primary antibody, is bound to the primary antibody. Colloidal gold particles are readily visible as dense spheres in electron microscope images. Colloidal gold labeling has been used to map Tetrahymena proteins in the oral apparatus, membrane skeleton, and nucleolus. For double labeling, two different primary antibodies are used followed by secondary antibodies labeled with gold particles of different size.

Chapter 19 Tetrahymena Telomerase Activity, Purification, and Reconstitution

Telomerase is a DNA replication enzyme responsible for the synthesis of telomeres, specific G-rich sequences at chromosome ends. A specific sequence within the RNA component of telomerase serves as a template for the synthesis of telomeric repeats, characterizing telomerase as a reverse transcriptase. Tetrahymena , like other ciliates such as Oxytricha and Euplotes , provides an abundant source of telomerase. The extensive chromosome fragmentation and the amplification that occur during macronuclear development in Tetrahymena result in the addition of telomeric repeats onto approximately 20,000 chromosome ends. Unlike other ciliates such as Oxytricha and Euplotes , Tetrahymena is cultured easily in defined media (10 5 cells/ml). In culturing Tetrahymena , soap residue left on glassware may inhibit cell growth. Thus, all glassware is kept soap free. All manipulations are performed using sterile techniques and sterile glassware, buffers, and media. Strains SB210 or CU428 are examples of strains routinely grown for preparation of Tetrahymena protein extracts. If the extract is prepared from mated cells, then two strains of different mating type are required.

An unusual fibrillarin gene and protein: structure and functional implications.

The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing. The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing. We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein. The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins. The remainder of the amino acid sequence of the molecule is more conserved. Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations. Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells. Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena. The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested.

Cultivar and processing effects on the pasting characteristics, tannin content and protein quality and digestibility of cowpea (Vigna unguiculata).

Four popular West African local cultivars of cowpea (Vigna unguiculata), with distinctly different seed coat colors, were evaluated for their relative amylograph pasting characteristics, condensed tannin content, in vitro protein digestibility and Tetrahymena protein efficiency ratio (t-PER). The effects of roasting and dehulling on these properties were also determined. There were wide variations in the hot paste viscosity characteristics of the different cultivars studied. The raw cowpea flour samples exhibited maximum paste viscosities ranging between 260 Brabender Units (BU) for the Mottled cultivar and 460 BU for the cream-colored Blackeye cultivar. Cowpea cultivars with the greatest peak viscosities showed low stabilities to extended cooking. Roasting depressed paste viscosity properties of all the cowpea cultivars studied. Tannin concentrations were 0.3-6.9 and 7.2-116 mg CE/g flour from whole cowpea seeds and seed coats respectively, increasing with intensity of seed color. Although dehulling removed 98% of the tannin content of raw cowpeas, improvement in protein quality as a result of dehulling was observed for only the highly-pigmented Maroon-red variety. Roasting significantly improved digestibility and more than doubled the t-PER of all cowpea cultivars studied. Roasted cowpeas possess adequate nutritional and functional qualities as protein supplements in cereal-based weaning foods. However, it appears that dehulling is necessary to enhance the nutritional quality of the highly pigmented cultivars of cowpea.