Christian Barth

Two Dictyostelium Orthologs of the Prokaryotic Cell Division Protein FtsZ Localize to Mitochondria and Are Required for the Maintenance of Normal Mitochondrial Morphology

ABSTRACT In bacteria, the protein FtsZ is the principal component of a ring that constricts the cell at division. Though all mitochondria probably arose through a single, ancient bacterial endosymbiosis, the mitochondria of only certain protists appear to have retained FtsZ, and the protein is absent from the mitochondria of fungi, animals, and higher plants. We have investigated the role that FtsZ plays in mitochondrial division in the genetically tractable protist Dictyostelium discoideum, which has two nuclearly encoded FtsZs, FszA and FszB, that are targeted to the inside of mitochondria. In most wild-type amoebae, the mitochondria are spherical or rod-shaped, but in fsz-null mutants they become elongated into tubules, indicating that a decrease in mitochondrial division has occurred. In support of this role in organelle division, antibodies to FszA and FszA-green fluorescent protein (GFP) show belts and puncta at multiple places along the mitochondria, which may define future or recent sites of division. FszB-GFP, in contrast, locates to an electron-dense, submitochondrial body usually located at one end of the organelle, but how it functions during division is unclear. This is the first demonstration of two differentially localized FtsZs within the one organelle, and it points to a divergence in the roles of these two proteins.

Chaperonin 60 and mitochondrial disease in Dictyostelium

The single Dictyostelium chaperonin 60 gene, hspA, was cloned, sequenced and characterized. Sequence comparisons and a three-dimensional model for the structure of the encoded protein showed that it exhibits the conserved sequence and structural features expected for its role as the Dictyostelium mitochondrial chaperonin 60. Dictyostelium hspA contains two introns and, unusually for a member of this major heat shock gene family, is not stress-inducible in response to heat, cold or cadmium ions. Although transcription of hspA is down regulated during early Dictyostelium development in response to starvation, the levels of the chaperonin 60 protein remain constant throughout the life cycle. Consistent with the essential role of chaperonin 60 in mitochondrial biogenesis, we were unable to isolate mutants in which the hspA gene had been disrupted. However, transformants were isolated that exhibited differing levels of antisense inhibition of chaperonin 60 expression, depending upon the number of copies of the antisense-expressing plasmid in the genome. Orientation in phototaxis (and thermotaxis) was severely impaired in all antisense transformants, while growth and morphogenesis were markedly defective only in transformants with higher levels of antisense inhibition. This pattern of phenotypes is similar to that reported previously to result from targeted disruption of the mitochondrial large subunit rRNA gene in a subpopulation of mitochondria. This suggests that, regardless of the nature of the underlying genetic defect, mitochondrial deficiency impairs signal transduction more sensitively than other cellular activities.

Mitochondrial biology and disease in Dictyostelium.

The cellular slime mold Dictyostelium discoideum has become an increasingly useful model for the study of mitochondrial biology and disease. Dictyostelium is an amoebazoan, a sister clade to the animal and fungal lineages. The mitochondrial biology of Dictyostelium exhibits some features which are unique, others which are common to all eukaryotes, and still others that are otherwise found only in the plant or the animal lineages. The AT-rich mitochondrial genome of Dictyostelium is larger than its mammalian counterpart and contains 56kb (compared to 17kb in mammals) encoding tRNAs, rRNAs, and 33 polypeptides (compared to 13 in mammals). It produces a single primary transcript that is cotranscriptionally processed into multiple monocistronic, dicistronic, and tricistronic mRNAs, tRNAs, and rRNAs. The mitochondrial fission mechanism employed by Dictyostelium involves both the extramitochondrial dynamin-based system used by plant, animal, and fungal mitochondria and the ancient FtsZ-based intramitochondrial fission process inherited from the bacterial ancestor. The mitochondrial protein-import apparatus is homologous to that of other eukaryote, and mitochondria in Dictyostelium play an important role in the programmed cell death pathways. Mitochondrial disease in Dictyostelium has been created both by targeted gene disruptions and by antisense RNA and RNAi inhibition of expression of essential nucleus-encoded mitochondrial proteins. This has revealed a regular pattern of aberrant mitochondrial disease phenotypes caused not by ATP insufficiency per se, but by chronic activation of the universal eukaryotic energy-sensing protein kinase AMPK. This novel insight into the cytopathological mechanisms of mitochondrial dysfunction suggests new possibilities for therapeutic intervention in mitochondrial and neurodegenerative diseases.

Polycistronic transcription and editing of the mitochondrial small subunit (SSU ) ribosomal RNA in Dictyostelium discoideum

Abstract Northern analyses and reverse transcription-polymerase chain reaction (RT-PCR) experiments were performed on total RNA of Dictyostelium discoideum. The mitochondrial genes encoding the small subunit ribosomal RNA (SSU), cytochrome b (CYTB) and subunit 3 of the NADH dehydrogenase (ND3) were found to be co-transcribed. Further post-transcriptional processing resulted in a dicistronic transcript for CYTB and ND3, and a monocistronic SSU transcript. Markedly higher steady state transcript levels were detected for the mature SSU ribosomal RNA. A comparison of the SSU cDNA sequence with the mitochondrial DNA sequence of the SSU gene revealed C-to-U substitutional editing of the SSU ribosomal RNA at a single site, as a consequence of which the cDNA contained a PvuII site not present in the genomic DNA. The editing was shown to be highly efficient and to occur in the primary transcript before the release of the mature mRNA, rRNA and tRNAs. It is suggested that the editing may be required for normal pseudoknot formation in the 530 loop of the RNA and thus is important for efficient, accurate translation in the mitochondria.

A rapid, small scale method for characterization of plasmid insertions in the Dictyostelium genome

A rapid, simple method for characterization of plasmid insertions in the Dictyostelium discoideum genome was developed. It is based on the capability of linear plasmid multimers in the insertions to recircularize efficiently in Escherichia coli cells. This recombinational recircularization of plasmid multimers provides a highly sensitive and reliable tool for determining whether individual Dictyostelium transformants resulted from restriction enzyme-mediated integration (REMI) or from recombinational integration of plasmid (RIP). The method also reveals any rearrangements in RIP insertions and provides an estimate of the vector copy number in any particular transformant.

Characterization of histamine projections and their potential cellular targets in the mouse retina

The vertebrate retina receives histaminergic input from the brain via retinopetal axons that originate from perikarya in the posterior hypothalamus. In the nervous system, histamine acts on three G-protein-coupled receptors, histamine receptor (HR) 1, HR2 and HR3. In order to look for potential cellular targets of histamine in the mouse retina, we have examined the retina for the expression of histamine and the presence of these three receptors. Consistent with studies of retina from other vertebrates, histamine was only found in retinopetal axons, which coursed extensively through the ganglion cell and inner plexiform layers. mRNA for all three receptors was expressed in the mouse retina, and immunohistochemical studies further localized HR1 and HR2. HR1 immunoreactivity was observed on dopaminergic amacrine cells, calretinin-positive ganglion cells and axon bundles in the ganglion cell layer. Furthermore, a distinct group of processes in the inner plexiform layer was labeled, which most likely represents the processes of cholinergic amacrine cells. HR2 immunoreactivity was observed on the processes and cell bodies of the primary glial cells of the mammalian retina, the Müller cells. This distribution of histamine and its receptors is consistent with a brain-derived source of histamine acting on diverse populations of cells in the retina, including both neurons and glia.

Identification of a novel pentatricopeptide repeat subfamily with a C-terminal domain of bacterial origin acquired via ancient horizontal gene transfer

BackgroundPentatricopeptide repeat (PPR) proteins are a large family of sequence-specific RNA binding proteins involved in organelle RNA metabolism. Very little is known about the origin and evolution of these proteins, particularly outside of plants. Here, we report the identification of a novel subfamily of PPR proteins not found in plants and explore their evolution.ResultsWe identified a novel subfamily of PPR proteins, which all contain a C-terminal tRNA guanine methyltransferase (TGM) domain, suggesting a predicted function not previously associated with PPR proteins. This group of proteins, which we have named the PPR-TGM subfamily, is found in distantly related eukaryotic lineages including cellular slime moulds, entamoebae, algae and diatoms, but appears to be the first PPR subfamily absent from plants. Each PPR-TGM protein identified is predicted to have different subcellular locations, thus we propose that these proteins have roles in tRNA metabolism in all subcellular locations, not just organelles. We demonstrate that the TGM domain is not only similar to bacterial TGM proteins, but that it is most similar to chlamydial TGMs in particular, despite the absence of PPR proteins in bacteria. Based on our data, we postulate that this subfamily of PPR proteins evolved from a TGM-encoding gene of a member of the Chlamydiae, which was obtained via ancient prokaryote-to-eukaryote horizontal gene transfer. Following its acquisition, the N-terminus of the encoded TGM protein must have been extended to include PPR motifs, possibly to confer additional functions to the protein, giving rise to the PPR-TGM subfamily.ConclusionsThe identification of a unique PPR subfamily which originated from the Chlamydiae group of bacteria offers novel insight into the origin and evolution of PPR proteins not previously considered. It also provides further understanding into their roles in non-organellar RNA metabolism.

A Unique Mitochondrial Transcription Factor B Protein in Dictyostelium discoideum

Unlike their bacteriophage homologs, mitochondrial RNA polymerases require the assistance of transcription factors in order to transcribe mitochondrial DNA efficiently. The transcription factor A family has been shown to be important for transcription of the human mitochondrial DNA, with some of its regulatory activity located in its extended C-terminal tail. The mitochondrial transcription factor B family often has functions not only in transcription, but also in mitochondrial rRNA modification, a hallmark of its α-proteobacterial origin. We have identified and characterised a mitochondrial transcription factor B homolog in the soil dwelling cellular slime mould Dictyostelium discoideum, an organism widely established as a model for studying eukaryotic cell biology. Using in bacterio functional assays, we demonstrate that the mitochondrial transcription factor B homolog not only functions as a mitochondrial transcription factor, but that it also has a role in rRNA methylation. Additionally, we show that the transcriptional activation properties of the D. discoideum protein are located in its extended C-terminal tail, a feature not seen before in the mitochondrial transcription factor B family, but reminiscent of the human mitochondrial transcription factor A. This report contributes to our current understanding of the complexities of mitochondrial transcription, and its evolution in eukaryotes.

Identification of Pentatricopeptide Repeat Proteins in the Model Organism Dictyostelium discoideum

Pentatricopeptide repeat (PPR) proteins are RNA binding proteins with functions in organelle RNA metabolism. They are found in all eukaryotes but have been most extensively studied in plants. We report on the identification of 12 PPR-encoding genes in the genome of the protist Dictyostelium discoideum, with potential homologs in other members of the same lineage and some predicted novel functions for the encoded gene products in protists. For one of the gene products, we show that it localizes to the mitochondria, and we also demonstrate that antisense inhibition of its expression leads to slower growth, a phenotype associated with mitochondrial dysfunction.

Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption.

BackgroundThe cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker.ResultsWe have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered.ConclusionsChanging the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain.