Dicty News Electronic Edition Volume 21, number 6 August 29, 2003 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Phosducin-like proteins in Dictyostelium discoideum: Implications for the phosducin family of proteins Mieke Blaauw, Jaco C. Knol, Arjan Kortholt, Jeroen Roelofs, Ruchira, Marten Postma, Antonie J.W.G. Visser and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, The Netherlands EMBO Journal, in press Retinal phosducin is known to sequester transducin Gbeta-gamma, thereby modulating transducin activity. Phosducin is a member of a family of phosducin-like proteins (PhLP) found in eukaryotes. Phylogeny of 33 phosducin-like proteins from metazoa, plants and lower eukaryotes identified three distinct groups named phosducin-I to -III. We discovered three phlp genes in Dictyostelium, each encoding a phosducin-like protein of a different group. Disruption of the phlp1 gene strongly impaired G protein signalling, apparently due to mislocalisation of Gbeta-gamma in phlp1-null cells. GFP-Gbeta and GFP-G-gamma are membrane associated in wild-type cells, but cytosolic in phlp1-null cells. Phlp2 disruption is lethal due to a synchronous collapse of the cells after 16-17 cell divisions. Phlp3 disruptants show no abnormal phenotype. These results establish a role of phosducin-like proteins in facilitating folding, localisation or function of proteins, in addition to modulating G protein signalling. Submitted by: P.J.M.van.Haastert [P.J.M.van.Haastert@chem.rug.nl] ------------------------------------------------------------------------------- Roc, a Ras/GTPase domain in complex proteins Leonard Bosgraaf and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, The Netherlands BBA, in press We identified a novel group of the Ras/GTPase superfamily, termed Roc, that is present as domain in complex proteins together with other domains, including Leucine rich repeats, Ankyrin repeats, WD40 repeats, kinase domains, RasGEF and RhoGAP domains. Roc is always succeeded by a novel 300-400 amino acids long domain, termed COR. Proteins with Roc/COR are present in prokaryotes, Dictyostelium, plant and metazoa. Submitted by: P.J.M.van.Haastert [P.J.M.van.Haastert@chem.rug.nl] ------------------------------------------------------------------------------- Chemoattractant-stimulated calcium influx in Dictyostelium discoideum does not depend on cGMP Douwe M. Veltman, Jan Sietse De Boer and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, The Netherlands BBA, in press Chemoattractant stimulation of Dictyostelium cells leads to the opening of calcium channels in the plasma membrane, causing extracellular calcium to flux into the cell. The genetically uncharacterised mutants stmF and KI8 show strongly altered chemoattractant stimulated cGMP responses. The aberrant calcium influx in these strains has provided evidence that the chemoattractant stimulated calcium influx is potentiated by cGMP. We have tested this hypothesis in genetically defined mutants by measuring the calcium influx in a strain that lacks intracellular cGMP due to the disruption of two guanylyl cyclases, and in a strain with increased cGMP levels caused by the disruption of two cGMP-degrading phosphodiesterases. The results reveal that the calcium influx stimulated by cAMP or folic acid is essentially identical in these strains. We conclude that cGMP is not involved in chemoattractant-stimulated calcium influx. Submitted by: P.J.M.van.Haastert [P.J.M.van.Haastert@chem.rug.nl] ------------------------------------------------------------------------------- Temporal and Spatial Expression of Ammonium Transporter Genes During Growth and Development of Dictyostelium discoideum. Susan C. Follstaedt, Janet H. Kirsten, Charles K. Singleton. Differentiation, in press Ammonia is an important signaling molecule involved in the regulation of development in Dictyostelium. During aggregation, ammonia gradients are established, and the ammonia concentration in the immediate environment or within a particular cell throughout development may vary. This is due to the rate of cellular ammonia production, its rate of loss by evaporation to the atmosphere or by diffusion into the substratum and perhaps to cellular transport by ammonia transporters (AMTs). Recent efforts in genome sequencing have identified three ammonia transporters in Dictyostelium. In addition to physically altering the levels of ammonia within cells, the AMTs also may play a role in ammonia signaling. As an initial step in identifying such a function, the temporal and spatial expression of the three amt genes is examined. RT-PCR demonstrates that each of the three amt mRNAs is present and relatively constant throughout growth and development. The spatial expression of these three amt genes is examined during multiple stages of Dictyostelium development using in situ hybridization. A distinct and dynamic pattern of expression is seen for the three genes. In general, amtA is expressed heavily in prestalk cells in a dynamic way, while amtB and amtC are expressed in prespore regions consistently throughout development. AmtC also is expressed in the most anterior tip of fingers and slugs, corresponding to cells that mediate ammonia?s effect on the choice between slug migration versus culmination. Indeed, amtC null cells have a slugger phenotype, suggesting AmtC functions in the signaling pathway underlying the mechanics of this choice. Submitted by: Charles Singleton [charle.k.singleton@vanderbilt.edu] =============================================================================== [End Dicty News, volume 21, number 6]