CSM News Electronic Edition Volume 6, number 6 March 16, 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 =========== Involvement of the Vacuolar Proton-translocating ATPase in Multiple Steps of the Endo-lysosomal System and in the Contractile Vacuole System of Dictyostelium discoideum Lesly A. Temesvari (1,2), Juan M. Rodriguez-Paris (1), John M. Bush (1,2,3), Linyi Zhang (1), and James A. Cardelli (1,2) (1) Department of Microbiology and Immunology and (2) Center for Excellence in Cancer Research Louisiana State University Medical Center Shreveport, LA 71130 (3) Present Address: Department of Biology University of Arkansas Little Rock, AR 72204 Journal of Cell Science, in press. Summary We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H+-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. CMA treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al., 1988 J. Biol. Chem. 264, 3454) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, a-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H+-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H+-ATPase. ------------------------------------------------------------------ Structures of Diphosphoinositol Pentakisphosphate and Bisdiphosphoinositol Tetrakisphosphate from Dictyostelium resolved by NMR Analysis Tim Laussmann, Reint Eujen, C. Michael Weisshuhn, Ulrich Thiel and Guenter Vogel* Wachbereich 9 - Chemie, Bergische Universitaet GHS Wuppertal, Gaussstrasse 20 D-42097 Wuppertal, FRG Biochem.J., in press Summary Diphospho-myo-inositol phosphates (PP-InsP5 and bis-PP-InsP4) were isolated from Dictyostelium in order to clarify the precise positional isomerism by two dimensional 1H-31P NMR analysis. The diphosphorylated inositol phosphates are 4-PP-Ins(1,2,3,5,6)P5 and 4,5-bis-PP-Ins(1,2,3,6)P4 or their corresponding enantiomers. The vicinal arrangement of the diphospho groups with its steric and electrostatic constraints possibly qualifies bis-PP-InsP4 as a metabolite with high phosphate group transfer potential in phosphotransferase reactions. ---------------------------------------------------------------------- How Slime Molds Evade Nematodes Richard H. Kessin, Gregg G. Gundersen, Victor Zaydfudim, Mark Grimson, and R. Lawrence Blanton Proceedings of the National Academy of Sciences, in press. Abstract: We have found a complex predator-prey association between the social amoeba Dictyostelium discoideum and the free soil living nematode Caenorhabditis elegans. C. elegans feeds on the amoebae and multiplies indefinitely when amoebae are the sole food source. In an environment created from soil, D. discoideum grows and develops, but not in the presence of C. elegans. During development, C. elegans feeds on amoebae until they aggregate and synthesize an extracellular matrix called the slime sheath. After t he sheath forms, the aggregate and slug are protected. Adult nematodes ingest Dictyostelium spores, which pass through the gut of the worm without loss of structure and remain viable. Nematodes kill the amoebae, but disperse the spores. The sheath that is constructed when the amoebae aggregate and the spore coats of the individual cells may protect against this predator. We also show that individual amoebae secrete compounds that repel nematodes. --------------------------------------------------------------------- A LYSOSOMAL CYSTEINE PROTEINASE FROM DICTYOSTELIUM DISCOIDEUM CONTAINS N-ACETYLGLUCOSAMINE-1-PHOSPHATE BOUND TO SERINE, BUT NOT MANNOSE-6-PHOSPHATE ON N-LINKED OLIGOSACCHARIDES. Darshini P. Mehta, Mie Ichikawa, Paramahans V. Salimath, James R. Etchison, Richard Haak, Adriana Manzi# and Hudson H. Freeze J. Biol. Chem., in press Summary Previous studies showed that vegetative Dictyostelium discoideum cells make a lysosomal proteinase, proteinase-1, that contains multiple GlcNAc-a-1-P residues in phosphodiester linkage to serine. In order to have a better insight to this protein and its unusual sugar modification, we purified proteinase-1 and examined its carbohydrate components by direct chemical analysis, enzymatic degradations, lectin affinity chromatography and carbohydrate specific antibodies. In contrast to earlier reports, we found that proteinase-1 contains 7.5 mole of Fuc, 8 mole of Man, 2 mole of Xyl and 30 moleGlcNAc per calculated mole of protein, but no Man-6-P residues were found. The protein binds to Concanavalin A and Wheat Germ Agglutinin lectin affinity columns, and PNGase-F digestion released most of the mannose and xylose, but only a small portion of the GlcNAc. b-elimination under reducing conditions released only GlcNAc-a-1-P as analyzed by fast atom bombardment mass spectrometry, but there was no evidence for the release of disaccharides or of fucitol. Rabbit antiserum and monoclonal antibodies prepared against proteinase-1 recognize GlcNAc-a-1-P residues in immunoblots and are specifically competed by UDP-GlcNAc or GlcNAc-a-1-P. Similar blots with other monoclonal antibodies showed the presence of mannose-6-sulfate on N-linked sugar chains, and a-fucose residues on the protein. Thus, proteinase-1 has at least two types of modifications: GlcNAc-a-1-P-Ser, which we call phosphoglycosylation, and N-linked oligosaccharides. This is the first purified lysosomal enzyme in Dictyostelium that does not contain Man-6-P residues. The GlcNAc-a-1-P specific antibodies also recognize a group of developmentally regulated proteins, especially enriched in vegetative cells. Some of them are also lysosomal cysteine proteinases, and all bind to the GlcNAc-a-1-P specific monoclonal antibody, but not to the mammalian CI-Man-6-P receptor. Conversely, lysosomal enzymes that have Man-6-P do not bind to the GlcNAc-a-1-P specific antibody. An exception to this is b-N-acetylglucosaminidase where 15% of the activity binds to this antibody. Thus, there appear to be two sets of lysosomal enzymes with distinctly different post-translational modifications. --------------------------------------------------------------------- [End CSM News, volume 6, number 6]