Dicty News Electronic Edition Volume 23, number 19 December 17, 2004 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Changes in Mg2+-Ion Concentration and Heavy Chain Phosphorylation Regulate the Motor Activity of a Class-I Myosin Setsuko Fujita-Becker, Ulrike Dürrwang§, Muriel Erent, Richard J. Clark, Michael A. Geeves, and Dietmar J. Manstein Institute for Biophysical Chemistry, OE 4350, Hannover Medical School, Carl-Neuberg-Straße 1, D-30623 Hannover, Germany and Department of Biosciences, University of Kent, Canterbury CT2 7NJ, United Kingdom Journal of Biological Chemistry, in press Class-I myosins are single-headed motor proteins, implicated in various motile processes including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass-1alpha, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS-site. TEDS-site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin-binding and the release of hydrolysis products. A surprising effect of Mg2+-ions on in vitro motility was discovered. Changes in the level of free Mg2+-ions, within the physiological range, are shown to modulate motor activity by inhibiting ADP-release. Our results indicate that higher concentrations of free Mg2+-ions stabilise the tension-bearing A.M.ADP state and shift the system from the production of rapid movement towards the generation of tension. Submitted by: Dietmar Manstein [manstein@bpc.mh-hannover.de] ----------------------------------------------------------------------------- Constitutively active G protein-coupled receptor mutants block Dictyostelium development Minghang Zhang, Mousumi Goswami, and Dale Hereld Department of Microbiology and Molecular Genetics, The University of Texas Medical School at Houston, Houston, Texas 77030, USA Molecular Biology of the Cell, in press cAR1, a G protein-coupled receptor (GPCR) for cAMP, is required for the multicellular development of Dictyostelium. The activation of multiple pathways by cAR1 is transient due to poorly defined adaptation mechanisms. To investigate this, we used a genetic screen for impaired development to isolate four dominant-negative cAR1 mutants, designated DN1-4. The mutant receptors inhibit multiple cAR1-mediated responses known to undergo adaptation. Reduced in vitro adenylyl cyclase activation by GTPgammaS suggests that they cause constitutive adaptation of this and perhaps other pathways. In addition, the DN mutants are constitutively phosphorylated, which normally requires cAMP binding, and possess cAMP affinities that are ~100-fold higher than that of wild-type cAR1. Two independent activating mutations, L100H and I104N, were identified. These residues occupy adjacent positions near the cytoplasmic end of the receptor's third transmembrane helix and correspond to the (E/D)RY motif of numerous mammalian GPCRs, which is believed to regulate their activation. Taken together, these findings suggest that the DN mutants are constitutively activated and block development by turning on natural adaptation mechanisms. Submitted by: Dale Hereld [dhereld@uth.tmc.edu] ----------------------------------------------------------------------------- Cyclic AMP Receptors of Dictyostelium Dale Hereld1 and Peter N. Devreotes2 1The University of Texas Health Science Center at Houston and 2The Johns Hopkins University In "Encyclopedia of Biological Chemistry" (W.J. Lennarz & M.D. Lane, eds.), Elsevier, Oxford, in press When confronted with starvation, the social amoeba Dictyostelium discoideum survives by undergoing multicellular development and sporulation. The coordination of these processes is achieved in part through intercellular communication using secreted adenosine 3',5'-cyclic monophosphate (cAMP) and a family of cell-surface cAMP receptors (cARs). The cARs are examples of G protein-coupled receptors (GPCRs), which enable eukaryotic cells in general to sense and respond to a wide array of environmental and hormonal signals ranging from single photons to large glycoprotein hormones. Due to their involvement in diverse physiological processes, GPCR-targeted drugs are frequently employed in medicine to treat many common conditions including inflammation, hypertension, heart failure, and neurologic and psychiatric disorders. Because GPCRs and the pathways they regulate are conserved in virtually all eukaryotes examined to date, genetically tractable microbes such as Dictyostelium have contributed significantly to our understanding of GPCR function and regulation. Submitted by: Dale Hereld [dhereld@uth.tmc.edu] ----------------------------------------------------------------------------- Dd-Alix, a conserved endosome-associated protein, controls Dictyostelium development Sara Mattei§, W. Jonathan Ryves+, Béatrice Blot*, Rémy Sadoul*, Adrian J. Harwood+, Michel Satre§, Gérard Klein§ and Laurence Aubry§ §Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR 5092 CNRS-CEA-UJF), DRDC, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France; +MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, UK; *Laboratoire Neurodégénérescence et Plasticité (EMI 0108 INSERM-UJF), CHU-Grenoble -BP 217, 38043 Grenoble Cedex 9, France accepted for publication in Developmental Biology We have characterized the Dictyostelium homolog of the mammalian protein Alix. Dd-Alix is encoded by a single gene and is expressed during vegetative growth and multicellular development. We showed that the alx null strain fails to complete its developmental program. Past the tight aggregate stage, morphogenesis is impaired, leading to markedly aberrant structures containing vacuolated and undifferentiated cells but no mature spores. The developmental defect is cell-autonomous as most cells remain of the PstB type even when mixed with wild-type cells. Complementation analysis with different Alix constructs allowed the identification of a 101-residue stretch containing a coiled-coil domain essential for Alix function. In addition, we showed that the protein associates in part with vesicular structures and that its distribution on a Percoll gradient overlaps that of the endocytic marker Vamp7. Dd-Alix also co-localizes with Dd-Vps32. In view of our data, and given the role of Vps32 proteins in membrane protein sorting and multivesicular body formation in yeast and mammals, we hypothesize that the developmental defects of the alx null strain result from abnormal trafficking of cell-surface receptors. Submitted by: Laurence Aubry [laubry@cea.fr] ============================================================================== [End Dicty News, volume 23, number 19]