CSM News Electronic Edition Volume 6, number 11 April 20, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== NDP Kinase Can Modulate Contraction of Dictyostelium Cytoskeletons Carmen Aguado-Velasco, Michel Veron1, Jo Ann Rambow, and Edward R. Kuczmarski Department of Physiology and Biophysics, Finch University of Health Sciences, The Chicago Medical School, North Chicago, IL 60064. 1Regulation Enzymatique des Activites Cellulaires, Institut Pasteur, 75724, Paris, Cedex 15, France Cell Motility and the Cytoskeleton, in press ABSTRACT Extraction of Dictyostelium amoebae with Triton X-100 produces robust cytoskeletons composed mainly of actin and myosin II. These cytoskeletons rapidly contract when mixed with Mg-ATP in simple buffers. The Triton-soluble fraction was found to contain a GTP-dependent activity that prevented contraction by Mg-ATP. This activity was purified, and identified as nucleoside diphosphate kinase (NDP kinase). The apparent inhibition resulted from pre-contraction of the cytoskeletons. Tightly bound cytoskeletal ADP was presumably phosphorylated, and the resulting ATP powered contraction. NDP kinase appeared to be unique in this capacity, since other regenerating systems did not cause pre-contraction. Reconstitution experiments demonstrated that the kinase must be in physical contact with the cytoskeleton. These results suggest that Dictyostelium NDP kinase is able to channel ATP to the myosin molecule, and this could play a role in directly regulating cytoskeletal contraction or in facilitating contraction under conditions where intracellular ATP concentrations are low. This ability to modulate cytoskeletal contraction could help to explain observations in other systems whereby defects in NDP kinase result in abnormal development or changes in the metastatic potential of cancer cells. ---------------------------------------------------------------------- Structure/function-studies on the pH-dependent actin-binding protein hisactophilin in Dictyostelium mutants Mechthild Stoeckelhuber *, Angelika A. Noegel @, Christoph Eckerskorn @ Jana Koehler @, Daniela Rieger * and Michael Schleicher * *Adolf-Butenandt-Institut / Zellbiologie, Ludwig-Maximilians-Universitaet, Schillerstr. 42, 80336 Muenchen @ Max-Planck-Institut fuer Biochemie, Am Klopferspitz 18a, 82151 Martinsried, Fed. Rep. of Germany J. Cell Sci., in press. Summary Our previous studies have shown that the actin-binding protein hisactophilin from Dictyostelium discoideum is a candidate for organizing the actin cytoskeleton at the plasma membrane in a pH-dependent manner. To further characterize this interaction we isolated hisactophilin overexpression (hisII+) and hisactophilin minus(his-) mutants. D. discoideum contains two hisactophilin isoforms, both genes are independently transcribed and carry a short intron at the same position of the coding region. The deduced amino acid sequence of hisactophilin II showed a characteristic high content of 35 histidine residues out of total 118 amino acids. After transformation of Dictyostelium AX2 wild-type cells with a genomic fragment designed to inactivate the hisactophilin I gene we obtained hisactophilin II overexpressing mutants (hisII+). Multiple integration of the vector led to strong overexpression of hisactophilin II which even outnumbered the actin concentration by a factor of two. Hisactophilin II protein showed the same biochemical properties as hisactophilin I during purification and in its pH-dependent binding to F-actin; as shown by mass spectrometry the hisactophilin II fraction was almost completely myristoylated despite of this high overexpression. The inactivation of both hisactophilin genes was achieved by gene replacement with a vector construct encompassing parts of gene I and gene II connected by a geneticin cassette. The properties of the hisII+ and his- cells with regard to growth in shaking culture and on Klebsiella plates, development, chemotaxis and morphology were not affected under normal conditions. However, the hisII+-transformants revealed a significant difference to wild-type cells and his- cells when the cytoplasmic pH was lowered by diethylstilbestrol (DES), a proton pump inhibitor. HisII+ cells were more resistant to the acidification; in contrast to AX2 wild-type cells and his- cells they did not form plasma membrane protrusions, showed an increase in F-actin content, and contained large clusters of F-actin. Lowering the internal pH caused an accumulation of hisactophilin below the plasma membrane. The fact that cells deficient in hisactophilin loose again the resistance to acidification is in good agreement with the hypothesis that hisactophilin functions as a pH-sensor at the plasma membrane by reversibly connecting the membrane with the actin cortical network upon local changes of the proton concentration. ---------------------------------------------------------------------- The aimless RasGEF is required for processing of chemotactic signals through G-protein coupled receptors in Dictyostelium Robert H. Insall*, Jane Borleis and Peter N. Devreotes Dept. of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA *MRC Laboratory for Molecular Cell Biology & Dept. of Physiology, University College London, Gower Street, London WC1E 6BT Current Biology, in press. Background Ras proteins are small GTP-binding proteins which are essential in a wide range of processes, in particular mammalian growth control. They act as molecular switches, being inactive when GDP is bound, and active when associated with GTP. Activation is accomplished by guanine nucleotide exchange factors (RasGEFs); when RasGEFs interact with Ras proteins, GDP is allowed to escape, and be replaced by GTP. Dictyostelium responds to chemoattractants through typical seven transmembrane domain receptors and heterotrimeric G-proteins. There are at least five different Dictyostelium Ras genes, whose functions are not yet known. Results We have isolated the aimless gene, which encodes the Dictyostelium homologue of Ras guanine nucleotide exchange factors (RasGEFs), during a screen for insertional mutants which fail to aggregate. Aimless null mutants grow at a normal rate, but are severely impaired in both chemotaxis and activation of adenylyl cyclase, which are both critical for the early stages of development. While coupling between receptors and their G-proteins is unaffected, and several cAMP-mediated responses appear normal, receptor and GTPgS mediated activation of adenylyl cyclase are reduced by up to 95%. The motility of mutant cells appears normal, suggesting a true defect in gradient sensing. Conclusions The discovery of the aimless gene adds an interesting new member to the family of RasGEFs. Our data suggest an unforeseen role for a RasGEF, and therefore presumably a complete Ras pathway, in the processing of chemotactic signals through G-protein coupled receptors. ---------------------------------------------------------------------- SP75 is encoded by the DP87 gene, and belongs to a family of modular Dictyostelium discoideum outer layer spore coat proteins Christopher M. West, Jie Mao, Hanke van der Wel, Gregory W. Erdos and Yunyan Zhang Department of Anatomy and Cell Biology, College of Medicine, and Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610-0235 USA Microbiology, in press SUMMARY Highly purified spore coats of Dictyostelium each contain about 5 X 10exp6 proteins as determined by amino acid composition analysis. By two-dimensional gel electrophoresis, the coat contains nine major abundance and numerous minor protein species, most of which are highly enriched relative to the adjacent interspore matrix. Protein is nearly quantitatively eluted by denaturants and 2-mercaptoethanol, showing that protein is not irreversibly cross-linked. Because a reducing agent is required together with denaturants to elute most proteins if their free sulfhydryl groups have been prealkylated, proteins appear to be disulfide cross-linked into the matrix. One major coat protein, SP75, was partially sequenced and found to be encoded by the previously identified DP87 gene; this conclusion is supported by additional physical, genetic, biochemical and microscopic evidence. The five major proteins for which genes have been cloned are associated with the outer layer of the coat. In coats which are missing one or more of four of these proteins as the result of gene disruption, there are physical changes but, with one exception, the other major coat proteins appear to be incorporated normally. Sequence analysis shows that these five outer layer coat proteins are homologous and consist of alternating sequence motifs related to epithelial mucin repeats, basic proline repeats found in salivary acidic proline-rich proteins, the NH2-terminal subdomain of EGF modules, and other cysteine repeats. Based on these and other observations, outer layer coat proteins are predicted to organize indeterminately to form a cell surface microenvironment supportive of cellulose morphogenesis during spore coat formation. -------------------------------------------------------------------- Purification and Characterization of an a1,2-L-Fucosyltransferase which Modifies the Cytosolic Protein FP21, from the Cytosol of Dictyostelium Christopher M. West, Toby Scott-Ward, Patana Teng-umnuay, Hanke van der Wel, Emil Kozarov, and Ann Huynh Department of Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235 USA J. Biol. Chem., in press SUMMARY A novel fucosyltransferase (cFTase) activity has been enriched over 10exp6-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galb1,3GlcNAcb-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent Mr of 95 x 103, was Mg+2-dependent with a neutral pH optimum, and exhibited a Km for GDP-fucose of 0.34 uM, a Km for pNP-LNB of 0.6 mM, and a Vmax for pNP-LNB of 620 nmol/min/mg protein. SDS-PAGE analysis of the Superdex elution profile identified a polypeptide with an apparent Mr of 85 x 10exp3, which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-[125I]salicylate. Based on substate analogue studies, exoglycosidase digestions, and cochromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fuca1,2Galb1,3GlcNAcb-pNP. The cFTase preferred substrates with a Galb1,3-linkage, and thus its acceptor substrate specificity resembles the human Secretor-type a1,2FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent Km of 0.21 uM and an apparent Vmax of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fuca1,2Galb1,3GlcNAcb-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations. -------------------------------------------------------------------- PsB multiprotein complex of Dictyostelium discoideum: Demonstration of cellulose binding activity and order of protein subunit assembly. Vince McGuire and Stephen Alexander Division of Biological Sciences, University of Missouri Columbia, MO 65211 J. Biol. Chem., in press. Abstract The differentiated spores of Dictyostelium are surrounded by an extracellular matrix (ECM) - the spore coat - which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique ECM is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, McGuire and Alexander, J. Cell Sci. 107, 2567-2579 (1994)). Later in development, the complex is synchronously secreted from the PSVs and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat. -------------------------------------------------------------------- [End CSM News, volume 6, number 11]