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Nested genes CDA12 and CDA13 encode proteins associated with membrane trafficking in the ciliate Tetrahymena thermophila.

ABSTRACT: We describe a novel pair of nested genes, CDA12 and CDA13, from Tetrahymena thermophila. Both are implicated in membrane trafficking associated with cell division and conjugation. Green fluorescent protein localization reveals Cda12p decoration of diverse membrane-bound compartments, including mobile, subcortical tubulovesicular compartments; perinuclear vesicles; and candidates for recycling endosomes. Cda13p decorates intracellular foci located adjacent to cortically aligned mitochondria and their neighboring Golgi networks. The expression of antisense CDA12 RNA in transformants produces defects in cytokinesis, macronuclear segregation, and the processing of pinosomes to downstream compartments. Antisense CDA13 RNA expression produces a conjugation phenotype, resulting in the failure of mating pairs to separate, as well as failures in postconjugation cytokinesis and macronuclear fission. This study offers insight into the membrane trafficking events linking endosome and Golgi network activities, cytokinesis, and karyokinesis and the unique membrane-remodeling events that accompany conjugation in the ciliate T. thermophila. We also highlight an unusual aspect of genome organization in Tetrahymena, namely, the existence of nested, antisense genes.

SUBMITTER: Zweifel E

PROVIDER: S-EPMC2698308 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Nested genes CDA12 and CDA13 encode proteins associated with membrane trafficking in the ciliate Tetrahymena thermophila.

Zweifel Erica E Smith Joshua J Romero Daniel D Giddings Thomas H TH Winey Mark M Honts Jerry J Dahlseid Jeff J Schneider Brent B Cole Eric S ES

Eukaryotic cell 20090313 6

We describe a novel pair of nested genes, CDA12 and CDA13, from Tetrahymena thermophila. Both are implicated in membrane trafficking associated with cell division and conjugation. Green fluorescent protein localization reveals Cda12p decoration of diverse membrane-bound compartments, including mobile, subcortical tubulovesicular compartments; perinuclear vesicles; and candidates for recycling endosomes. Cda13p decorates intracellular foci located adjacent to cortically aligned mitochondria and t ...[more]

PMID: 19286988

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Project description:The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.

Project description:BackgroundTetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.ResultsFunctional and full length human intestinal alkaline phosphatase was expressed by T. thermophila using a codon-adapted gene containing the native signal-peptide and GPI (Glycosylphosphatidylinositol) anchor attachment signal. HiAP activity in the cell extract of transformants suggested that the hiAP gene was successfully expressed. Furthermore, it was demonstrated that the enzyme was modified with N-glycosylation and localized on the surface membrane by the C-terminal GPI anchor. A C-terminally truncated version of hiAP lacking the GPI anchor signal peptide was secreted into the medium as an active enzyme. In a first approach to establish a high level expression system up to 14,000 U/liter were produced in a time frame of two days, which exceeds the production rate of other published expression systems for this enzyme.ConclusionsWith the expression of hiAP, not only a protein of commercial interest could be produced, but also a reporter enzyme that offers the possibility to analyze T. thermophila genes that play a role in the regulation of protein secretion. Additionally, the fact that ciliates do not secrete an endogenous alkaline phosphatase provides the possibility to use the truncated hiAP as a reporter enzyme, allowing the quantification of measures that will be necessary for further optimization of the host strains and the fermentation processes.

Dataset Information

Nested genes CDA12 and CDA13 encode proteins associated with membrane trafficking in the ciliate Tetrahymena thermophila.

Publications

Nested genes CDA12 and CDA13 encode proteins associated with membrane trafficking in the ciliate Tetrahymena thermophila.

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