Dicty News Electronic Edition Volume 17, number 10 October 27, 2001 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 ============= The NF-kB like DNA binding activity observed in Dictyostelium nuclear extracts is due to the GBF transcription factor Franois TRAINCARD 1, Eleonora PONTE 1, Jason PUN 2, Barrie COUKELL 2 and Michel VERON 1 1: Unit de Rgulation enzymatique des Activits cellulaires, CNRS FRE 2364, Institut Pasteur, 25 rue du Dr. Roux, 75724, Paris Cedex 15, France. 2: York University, 4700 Keele St., Toronto, Ontario, M3J 1P3, Canada. Accepted in Journal of Cell Science We have previously reported that a NF-kB transduction pathway was likely to be present in the cellular slime mold Dictyostelium discoideum (Traincard, F., Ponte, E., Pun, J., Coukell, B. and Vron, M., 1999, J. Cell Science, 112, 3529-3535). This conclusion was based on several observations, including the detection of developmentally regulated DNA binding proteins in Dictyostelium nuclear extracts, which bound to bona fide kB sequences. We have now performed additional experiments that demonstrate that the protein responsible for this NF-kB like DNA binding activity is the Dictyostelium GBF (G box regulatory element Binding Factor) transcription factor. This result, along with the fact that no sequence with significant similarity to components of the mammalian NF-kB pathway can be found in Dictyostelium genome, now almost entirely sequenced, led us to reconsider our previous conclusion on the occurrence of a NF-kB signal transduction pathway in Dictyostelium. ----------------------------------------------------------------------------- GTP[gamma]S regulation of a twelve transmembrane guanylyl cyclase is retained after mutation to an adenylyl cyclase Jeroen Roelofs, Harrit M. Loovers and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4,9747AG Groningen, the Netherlands J. Biol. Chem., in press ABSTRACT DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing twelve transmembrane spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extra-cellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by GTPgS, which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCAkqd in adenylyl cyclase defective cells restored the phenotype of the mutant. GTPgS stimulated the adenylyl cyclase activity of DdGCAkqd with similar properties as the wildtype enzyme (decrease of Km and increase of Vmax). This demonstrates that GTPgS-stimulation is independent of substrate specificity. Furthermore, GTPgS- activation of DdGCAkqd is retained in several null mutants of Ga and Gb proteins, indicating that GTPgS-activation is not mediated by a heterotrimeric G-protein, but possibly by a monomeric G-protein. ----------------------------------------------------------------------------- Single molecule analysis of chemotactic signaling in Dictyostelium cells Masahiro Ueda, Yasushi Sako, Toshiki Tanaka, Peter Devreotes & Toshio Yanagida Science 294, 864-867. Single-molecule imaging techniques were used to reveal the binding of individual cyclic adenosine 3',5'-monophosphate molecules to heterotrimeric guanine nucleotide-binding protein coupled receptors on the surface of living Dictyostelium cells. The binding sites were uniformly distributed and diffused rapidly in the plane of the membrane. The probabilities of individual association and dissociation events were greater for receptors at the anterior end of the cell. Agonist-induced receptor phosphorylation had little effect on any of the monitored properties, whereas G-protein coupling influenced the binding kinetics. These observations illustrate the dynamic properties of receptors involved in gradient sensing and suggest that these may be polarized in chemotactic cells. ----------------------------------------------------------------------------- Calreticulin and Calnexin in the Endoplasmic Reticulum Are Important for Phagocytosis Annette Mueller-Taubenberger*, Andrei N. Lupas, Hewang Li*, Mary Ecke*, Evelyn Simmeth*, and Guenther Gerisch* * Max-Planck-Institut fuer Biochemie, Martinsried, Germany SmithKline Beecham Pharmaceuticals UP1345, Collegeville, PA, USA EMBO Journal, in press. Abstract Calreticulin and calnexin are Ca2+-binding proteins with chaperone activity in the endoplasmic reticulum. These proteins have been eliminated by gene replacement in Dictyostelium, the only microorganism known to harbor both proteins; family members in Dictyostelium are located at the base of phylogenetic trees. In double mutants lacking calreticulin and calnexin a dramatic decline in the rate of phagocytosis was observed, whereas only mild changes occurred in single mutants. Dictyostelium cells are professional phagocytes, capable of internalizing particles by a sequence of activities: adhesion of the particle to the cell surface, actin- dependent outgrowth of a phagocytic cup, and separation of the phagosome from the plasma membrane. In the double-null mutants, particles still adhered to the cell surface, but the outgrowth of phagocytic cups was compromised. GFP-tagged calreticulin and calnexin, expressed in wild type cells, revealed a direct link of the ER to the phagocytic cup enclosing a particle, such that the Ca2+-storage capacity of calreticulin and calnexin might directly modulate activities of the actin system during particle uptake. ----------------------------------------------------------------------------- [End Dicty News, volume 17, number 10]