CSM News Electronic Edition Volume 2, number 22 June 18, 1994 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. =========== Abstracts =========== A Rab4-like GTPase in Dictyostelium discoideum Colocalizes with V-H+-ATPases in Reticular Membranes of the Contractile Vacuole Complex and in Lysosomes John Bush*, Kathleen Nolta^, Juan Rodriguez-Paris*, Nancy Kaufmann#, Terry O'Halloran#, Tracy Ruscetti*, Lesly Temesvari*, Theodore Steck^, and James Cardelli* *Departments of Microbiology and Immunology, LSU Medical Center, Shreveport, LA; ^Biochemistry and Molecular Biology, Univ. of Chicago, Chicago, IL and #Cell Biology, Duke University Medical Center, Durham, NC. Journal of Cell Science, in press In the course of screening a cDNA library for ras-related D. discoideum genes, we cloned a 0.7 kB cDNA (rabD) encoding a putative protein that was 70% identical at the amino acid level to human Rab4. Rab4 is a small GTPase that belongs to the Ras superfamily and functions to regulate endocytosis in mammalian cells. Southern blot analysis indicated that the rabD cDNA was encoded by a single copy gene while Northern blots revealed that the RabD gene was expressed at relatively constant levels during growth and differentiation. Affinity purified antibodies were prepared against a RabD fusion protein expressed in bacteria; the antibodies recognzied a single 23 kDa protein on Western blots of cell extracts. Density gradient fractionation revealed that the RabD antigen co-distributed primarily with bouyant membranes rich in vacuolar proton pumps and to a lesser extent with lysosomes. This result was confirmed by examining celll lines expressing an epitope tagged version of RabD. Magnetically purified early endocytic vesicles and postlysosomal vacuoles reacted more weakly with the anti-RabD antibodies than did lysosomes. Other organelles were negative for RabD. Double label indirect IF microscopy revealed that RabD and the 100 kDa proton pump subunit colocalized in a fine reticular network throughout the cytoplasm. This network was reminiscent of spongiomes, the tubular elements of the contractile vacuole system. Immunoelectron microscopy confirmed the presence of RabD in lysosome fractions and in the membranes rich in proton pumps. We conclude that a Rab4-like GTPase in D. discoideum is principally associated with the spongiomes of the contractile vacuole system. ------------------------------------------------------------------- Specific Induction by Zinc of Dictyostelium Stalk Cell Differentiation Yuzuru Kubohara# and Koji Okamoto #Department of Physiology, Institute of Endocrinology, Gunma University, Maebashi 371 Japan and Department of Botany, Faculty of Science, Kyoto University, Kyoto, 606-01 Japan Exp. Cell Res., in press. Summary Zinc is found in a variety of organisms and has been suggested to be essential for many cellular functions. We report here that zinc ions (Zn2+) have a specific and striking effect on cell differentiation in the simple eukaryote, Dictyostelium discoideum. In its asexual reproduction phase, solitary cells gather to form multicellular fruiting bodies consisting of spores and stalk cells. In vitro studies have revealed endogenous factors required for the differentiation of these two cell types. For stalk cell differentiation, these are cyclic AMP and a putative morphogen, a unique chlorinated alkylphenone named differentiation inducing factor (DIF). With millimolar concentrtions of Zn2+, however, cells neglect these requirements altogether and differentiate in vitro into mature stalk cells without the addition of cAMP and/or DIF, expressing stalk specific markers, wst34 (protein) and ecmB (gene), but not ecmA. Zn2+-induced stalk cell formation is thus distinct from normal stalk cell formation and this system would provide a clue to elucidate the mechanism of stalk cell differentiation. ----------------------------------------------------------------------- Two cAMP Receptors Activate Common Signalling Pathways in Dictyostelium Robert H. Insall, Ron D. M. Soede*, Pauline Schaap* and Peter N. Devreotes Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA *Department of Biology, University of Leiden, Wassenaarsweg 64, 2333 AL Leiden, The Netherlands Mol. Biol. Cell, in press. Abstract Multiple signal transduction pathways within a single cell may share common components. In particular, different seven transmembrane helix receptors may activate identical pathways by interacting with the same G-proteins. Dictyostelium cells respond to cAMP using one such receptor, cAR1, coupled by a typical heterotrimeric G-protein to intracellular effectors. However, cells in which the gene for cAR1 has been deleted are unexpectedly still able to respond to cAMP (Pupillo et al., 1992) . This implies either that certain responses are mediated by a different receptor than cAR1, or alternatively that a second, partially redundant receptor shares some of the functions of cAR1. We have examined the dose-response and ligand specificity of one response, cAMP relay, and the dose response of another, cyclic GMP synthesis. In each case, the EC50 was approximately 100-fold higher and the maximal response was smaller in car1- than wild-type cells. These data indicate that cAR1 normally mediates responses to cAMP. The ligand specificity suggests that the responses seen in car1- mutants are mediated by a second receptor, cAR3. To test this hypothesis, we constructed a cell line containing deletions of both cAR1 and cAR3 genes. As predicted, these lines are totally insensitive to cAMP. We conclude that the functions of the cAR1 and cAR3 receptors are partially redundant, and that both interact with the same same heterotrimeric G-protein to mediate these and other responses. ------------------------------------------------------------------- CRAC, a Cytosolic Protein Containing a Pleckstrin Homology Domain, is Required for Receptor and G-Protein Mediated Activation of Adenylyl Cyclase in Dictyostelium Robert Insall1, Adam Kuspa2*, Pamela J. Lilly1, Gad Shaulsky2, Lonny R. Levin3, William F. Loomis2, and Peter Devreotes1 1Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21205 2Center for Molecular Genetics, Department of Biology, University of California San Diego, La Jolla, CA 92093 3Howard Hughes Medical Institute, School of Medicine, Johns Hopkins University, Baltimore, MD 21205 *Present Address: Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 J. Cell Biol., in press. Summary Adenylyl cyclase in Dictyostelium, as in higher eukaryotes, is activated through G-protein coupled receptors. Insertional mutagenesis into a gene designated dagA resulted in cells that cannot activate adenylyl cyclase, but have otherwise normal responses to exogenous cAMP. Neither cAMP treatment of intact cells nor GTPgS treatment of lysates stimulates adenylyl cyclase activity in dagA mutants. A cytosolic protein which activates adenylyl cyclase, CRAC, has been previously identified. We trace the signalling defect in dagA- cells to the absence of CRAC, and we demonstrate that dagA is the structural gene for CRAC. The 3.2kb dagA mRNA encodes a predicted 78.5 kDa product containing a pleckstrin homology (PH) domain, in agreement with the postulated interaction of CRAC with activated G-proteins. Although dagA expression is tightly developmentally regulated, the cDNA restores normal development when constitutively expressed in transformed mutant cells. In addition, the megabase region surrounding the dagA locus was mapped. We hypothesize that CRAC acts to connect free G-protein bg-subunits to adenylyl cyclase activation. If so, it may be the first member of an important class of coupling proteins. --------------------------------------------------------------------- [End CSM News, volume 2, number 22]