Characterization of the Roco protein family in Dictyostelium discoideum.
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ABSTRACT:
SUBMITTER: van Egmond WN
PROVIDER: S-EPMC2863948 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:GbpC is a multidomain Roco protein in Dictyostelium, involved in transduction of intracellular cGMP that is produced by chemotactic signals. We have shown previously that cGMP binding to GbpC induces an intramolecular signaling cascade by activating subsequently the GEF, Ras, and kinase domains. In this study, we report on the cellular localization of GbpC. In resting cells, the protein is present in the cytoplasm, but GbpC rapidly translocates to the cell boundary upon stimulation with the chemoattractant cAMP. Also, during the formation of cell-cell streams and osmotic shock, the protein localizes toward the plasma membrane and actin cytoskeleton. The translocation upon cAMP stimulation occurs downstream of heterotrimeric G proteins but is independent of guanylyl cyclases and the previously identified cGMP-induced intramolecular signaling cascade in GbpC. Mutations in the GRAM domain of GbpC lead to disturbed membrane association and inactivation of GbpC function during chemotaxis in vivo. Furthermore, we show that the GRAM domain itself associates with cellular membranes and binds various phospholipids in vitro. Together, the results show that GbpC receives multiple input signals that are both required for functional activity in vivo. cAMP-stimulation induces a cGMP-dependent signaling cascade, leading to activation of kinase activity, and, independently, cAMP induces a GRAM-dependent translocation of GbpC toward the plasma membrane and cell cortex, where it may locally phosphorylate effector proteins, which are needed for proper biological activity.
| S-EPMC3268432 | biostudies-literature
Project description:Taking advantage of the ongoing Dictyostelium genome sequencing project, we have assembled >73 kb of genomic DNA in 15 contigs harbouring 15 genes and one pseudogene of Rho-related proteins. Comparison with EST sequences revealed that every gene is interrupted by at least one and up to four introns. For racC extensive alternative splicing was identified. Northern blot analysis showed that mRNAs for racA, racE, racG, racH and racI were present at all stages of development, whereas racJ and racL were expressed only at late stages. Amino acid sequences have been analysed in the context of Rho-related proteins of other organisms. Rac1a/1b/1c, RacF1/F2 and to a lesser extent RacB and the GTPase domain of RacA can be grouped in the Rac subfamily. None of the additional Dictyostelium Rho-related proteins belongs to any of the well-defined subfamilies, like Rac, Cdc42 or Rho. RacD and RacA are unique in that they lack the prenylation motif characteristic of Rho proteins. RacD possesses a 50 residue C-terminal extension and RacA a 400 residue C-terminal extension that contains a proline-rich region, two BTB domains and a novel C-terminal domain. We have also identified homologues for RacA in Drosophila and mammals, thus defining a new subfamily of Rho proteins, RhoBTB.
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Project description:Autophagy is a highly conserved intracellular degradative pathway that is crucial for cellular homeostasis. During autophagy, the core autophagy protein ATG12 plays, together with ATG5 and ATG16, an essential role in the expansion of the autophagosomal membrane. In this study we analyzed gene replacement mutants of atg12 in Dictyostelium discoideum AX2 wild-type and ATG16? cells. RNAseq analysis revealed a strong enrichment of, firstly, autophagy genes among the up-regulated genes and, secondly, genes implicated in cell motility and phagocytosis among the down-regulated genes in the generated ATG12?, ATG16? and ATG12?/16? cells. The mutant strains showed similar defects in fruiting body formation, autolysosome maturation, and cellular viability, implying that ATG12 and ATG16 act as a functional unit in canonical autophagy. In contrast, ablation of ATG16 or of ATG12 and ATG16 resulted in slightly more severe defects in axenic growth, macropinocytosis, and protein homeostasis than ablation of only ATG12, suggesting that ATG16 fulfils an additional function in these processes. Phagocytosis of yeast, spore viability, and maximal cell density were much more affected in ATG12?/16? cells, indicating that both proteins also have cellular functions independent of each other. In summary, we show that ATG12 and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy.
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Project description:Phagocytic cells ingest and destroy bacteria efficiently and in doing so ensure the defense of the human body against infections. Phagocytic Dictyostelium discoideum amoebae represent a powerful model system to study the intracellular mechanisms ensuring destruction of ingested bacteria in phagosomes. Here, we discovered the presence of a bacteriolytic activity against Klebsiella pneumoniae in cellular extracts from D. discoideum. The bacteriolytic activity was detected only at a very acidic pH mimicking the conditions found in D. discoideum phagosomes. It was also strongly decreased in extracts of kil1 KO cells that were previously described to kill inefficiently internalized bacteria, suggesting that the activity observed in vitro is involved in killing of bacteria in phagosomes. We purified a fraction enriched in bacteriolytic activity where only 16 proteins were detected and focused on four proteins selectively enriched in this fraction. Three of them belong to a poorly characterized family of D. discoideum proteins exhibiting a DUF3430 domain of unknown function and were named BadA (Bacteriolytic D. discoideum A), BadB, and BadC. We overexpressed the BadA protein in cells, and the bacteriolytic activity increased concomitantly in cell extracts. Conversely, depletion of BadA from cell extracts decreased significantly their bacteriolytic activity. Finally, in cells overexpressing BadA, bacterial killing was faster than in parental cells. Together these results identify BadA as a D. discoideum protein required for cellular bactericidal activity. They also define a new strategy to identify and characterize bactericidal proteins in D. discoideum cells.
| S-EPMC7886984 | biostudies-literature
Project description:BACKGROUND: Peptide: N- glycanase (PNGase) enzyme cleaves oligosaccharides from the misfolded glycoproteins and prepares them for degradation. This enzyme plays a role in the endoplasmic reticulum associated degradation (ERAD) pathway in yeast and mice but its biological importance and role in multicellular development remain largely unknown. RESULTS: In this study, the PNGase from the cellular slime mold, Dictyostelium discoideum (DdPNGase) was identified based on the presence of a common TG (transglutaminase) core domain and its sequence homology with the known PNGases. The domain architecture and the sequence comparison validated the presence of probable functional domains in DdPNGase. The tertiary structure matched with the mouse PNGase. Here we show that DdPNGase is an essential protein, required for aggregation during multicellular development and a knockout strain of it results in small sized aggregates, all of which did not form fruiting bodies. The in situ hybridization and RT-PCR results show higher level of expression during the aggregate stage. The expression gets restricted to the prestalk region during later developmental stages. DdPNGase is a functional peptide:N-glycanase enzyme possessing deglycosylation activity, but does not possess any significant transamidation activity. CONCLUSIONS: We have identified and characterized a novel PNGase from D. discoideum and confirmed its deglycosylation activity. The results emphasize the importance of PNGase in aggregation during multicellular development of this organism.
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Project description:Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce D-threo-BH(4) [6R-(1'R,2'R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of L-erythro-BH(4), in the last step of tetrahydrobiopterin (BH(4)) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH(4). To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR-NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 x 0.6 x 0.1 mm. The crystal belonged to space group P2(1), with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 A, beta = 100.00 degrees , and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR-NAD dimers. Diffraction data were collected to 2.16 A resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method.
| S-EPMC2581689 | biostudies-literature
Project description:The cell cycle phase at starvation influences post-starvation differentiation and morphogenesis in Dictyostelium discoideum. We found that when expressed in Saccharomyces cerevisiae, a D. discoideum cDNA that encodes the ribosomal protein S4 (DdS4) rescues mutations in the cell cycle genes cdc24, cdc42 and bem1. The products of these genes affect morphogenesis in yeast via a coordinated moulding of the cytoskeleton during bud site selection. D. discoideum cells that over- or under-expressed DdS4 did not show detectable changes in protein synthesis but displayed similar developmental aberrations whose intensity was graded with the extent of over- or under-expression. This suggested that DdS4 might influence morphogenesis via a stoichiometric effect--specifically, by taking part in a multimeric complex similar to the one involving Cdc24p, Cdc42p and Bem1p in yeast. In support of the hypothesis, the S. cerevisiae proteins Cdc24p, Cdc42p and Bem1p as well as their D. discoideum cognates could be co-precipitated with antibodies to DdS4. Computational analysis and mutational studies explained these findings: a C-terminal domain of DdS4 is the functional equivalent of an SH3 domain in the yeast scaffold protein Bem1p that is central to constructing the bud site selection complex. Thus in addition to being part of the ribosome, DdS4 has a second function, also as part of a multi-protein complex. We speculate that the existence of the second role can act as a safeguard against perturbations to ribosome function caused by spontaneous variations in DdS4 levels.
| S-EPMC3281849 | biostudies-literature
Project description:The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive alpha-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 micron/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.
| S-EPMC25463 | biostudies-literature
Project description: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.
| S-EPMC3724811 | biostudies-literature
Project description:Upon starvation, Dictyostelium discoideum cells halt cell proliferation, aggregate into multicellular organisms, form migrating slugs, and undergo morphogenesis into fruiting bodies while differentiating into dormant spores and dead stalk cells. At almost any developmental stage cells can be forced to dedifferentiate when they are dispersed and diluted into nutrient broth. However, migrating slugs can traverse lawns of bacteria for days without dedifferentiating, ignoring abundant nutrients and continuing development. We now show that developing Dictyostelium cells revert to the growth phase only when bacteria are supplied during the first 4 to 6 h of development but that after this time, cells continue to develop regardless of the presence of food. We postulate that the cells' inability to revert to the growth phase after 6 h represents a commitment to development. We show that the onset of commitment correlates with the cells' loss of phagocytic function. By examining mutant strains, we also show that commitment requires extracellular cyclic AMP (cAMP) signaling. Moreover, cAMP pulses are sufficient to induce both commitment and the loss of phagocytosis in starving cells, whereas starvation alone is insufficient. Finally, we show that the inhibition of development by food prior to commitment is independent of contact between the cells and the bacteria and that small soluble molecules, probably amino acids, inhibit development during the first few hours and subsequently the cells become unable to react to the molecules and commit to development. We propose that commitment serves as a checkpoint that ensures the completion of cooperative aggregation of developing Dictyostelium cells once it has begun, dampening the response to nutritional cues that might inappropriately block development.
| S-EPMC2168402 | biostudies-literature