Christophe Anjard

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal–fungal lineage after the plant–animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Unconventional secretion of Acb1 is mediated by autophagosomes

Evidence is presented for an unconventional protein secretion pathway that is conserved from yeast to Dictyostelium discoideum in which Acb1 may be sequestered into autophagosomal vesicles, which then fuse (either directly or indirectly) with the plasma membrane (see also the companion paper from Manjithaya et al. in this issue).

Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation

Evidence is presented for an unconventional protein secretion pathway that is conserved from yeast to Dictyostelium discoideum in which Acb1 may be sequestered into autophagosomal vesicles, which then fuse (either directly or indirectly) with the plasma membrane (see also the companion paper from Duran et al. in this issue).

The Golgi-Associated Protein GRASP Is Required for Unconventional Protein Secretion during Development

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.

Comparative genomics of the social amoebae Dictyostelium discoideum and Dictyostelium purpureum

BackgroundThe social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum.ResultsWe have produced a draft genome sequence of another group dictyostelid, Dictyosteliumpurpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict.ConclusionsThe findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia.

SDF-2 Induction of Terminal Differentiation in Dictyostelium discoideum Is Mediated by the Membrane-Spanning Sensor Kinase DhkA

ABSTRACT SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate. Genetic evidence indicates that the response is dependent on the dhkA gene. This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways. The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310-amino-acid loop that could bind a ligand. By inserting MYC6 epitopes into DhkA, we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic. Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100-fold-higher levels of SDF-2 than required to inducedhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope. To examine the enzymatic activity of DhkA, we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation. Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation. Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity. Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB. Suppressor studies indicate that the cyclic AMP (cAMP) phosphodiesterase RegA and the cAMP-dependent protein kinase PKA act downstream of DhkA.

Evolutionary analyses of ABC transporters of Dictyostelium discoideum.

ABSTRACT The ABC superfamily of genes is one of the largest in the genomes of both bacteria and eukaryotes. The proteins encoded by these genes all carry a characteristic 200- to 250-amino-acid ATP-binding cassette that gives them their family name. In bacteria they are mostly involved in nutrient import, while in eukaryotes many are involved in export. Seven different families have been defined in eukaryotes based on sequence homology, domain topology, and function. While only 6 ABC genes in Dictyostelium discoideum have been studied in detail previously, sequences from the well-advanced Dictyostelium genome project have allowed us to recognize 68 members of this superfamily. They have been classified and compared to animal, plant, and fungal orthologs in order to gain some insight into the evolution of this superfamily. It appears that many of the genes inferred to have been present in the ancestor of the crown organisms duplicated extensively in some but not all phyla, while others were lost in one lineage or the other.

GABA induces terminal differentiation of Dictyostelium through a GABAB receptor

When prespore cells approach the top of the stalk in a Dictyostelium fruiting body, they rapidly encapsulate in response to the signalling peptide SDF-2. Glutamate decarboxylase, the product of the gadA gene, generates GABA from glutamate. gadA is expressed exclusively in prespore cells late in development. We have found that GABA induces the release of the precursor of SDF-2, AcbA, from prespore cells. GABA also induces exposure of the protease domain of TagC on the surface of prestalk cells where it can convert AcbA to SDF-2. The receptor for GABA in Dictyostelium, GrlE, is a seven-transmembrane G-protein-coupled receptor that is most similar to GABAB receptors. The signal transduction pathway from GABA/GrlE appears to be mediated by PI3 kinase and the PKB-related protein kinase PkbR1. Glutamate acts as a competitive inhibitor of GABA functions in Dictyostelium and is also able to inhibit induction of sporulation by SDF-2. The signal transduction pathway from SDF-2 is independent of the GABA/glutamate signal transduction pathway, but the two appear to converge to control release of AcbA and exposure of TagC protease. These results indicate that GABA is not only a neurotransmitter but also an ancient intercellular signal.

Unconventional Secretion of AcbA in Dictyostelium discoideum through a Vesicular Intermediate

ABSTRACT The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.

Cytokinins induce sporulation in Dictyostelium.

The social amoeba Dictyostelium discoideum diverged from the line leading to animals shortly after the separation of plants and animals but it retained characteristics of both kingdoms. A GABAB-like receptor and a peptide, SDF-2, with homologs found only in animals, control sporulation, while cytokinins, which act as hormones in plants, keep spores dormant. When SDF-2 binds its receptor DhkA, it reduces the activity of the cAMP phosphodiesterase RegA such that cAMP levels can increase. It has been proposed that the cytokinin discadenine also produces in an increase in cAMP but acts through a different histidine kinase, DhkB. We have found that discadenine and its precursor, isopentenyl adenine, not only maintain spore dormancy but also initiate rapid encapsulation independently of the SDF-2 signal transduction pathway. DhkB and the adenylyl cyclase of late development, AcrA, are members of two component signal transduction families and both are required to transduce the cytokinin signal. As expected, strains lacking the isopentenyl-transferase enzyme chiefly responsible for cytokinin synthesis are defective in sporulation. It appears that SDF-2 and cytokinins are secreted during late development to trigger signal transduction pathways that lead to an increase in the activity of the camp-dependent protein kinase, PKA, which triggers rapid encapsulation as well as ensuring spore dormancy.