CSM News Electronic Edition Volume 8, number 4 February 1, 1997 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 =========== Trehalase of Dictyostelium discoideum: Inhibition by Amino-containing Analogs of Trehalose and Affinity Purification L.A. Temesvari (1,2) and D.A. Cotter (1) (1) Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada N9B 3P4. (2 Present Address) Department of Microbiology and Immunology and Center for Excellence in Cancer Research, Louisiana State University Medical Center, Shreveport, LA 71130. Biochimie, in press. Summary The inhibitory effects of three nitrogen-containing analogs of trehalose, Validamycin A, MDL 25,637 and castanospermine, on Dictyostelium discoideum trehalase were examined. Prior to this study, the effects of glycohydrolase inhibitors on D. discoideum trehalase have not been reported. Validamycin A, MDL 25,637 and castanospermine were found to be potent, reversible, competitive inhibitors of D. discoideum vegetative trehalase in vitro with IC50 values of 1 X 10-9 M, 2 X 10-8 M and 1.25 X 10-4 M, respectively. Validamycin A and MDL 25,637 also exhibited time dependent inhibition of D. discoideum trehalase, whereby the potencies of these two inhibitors were observed to increase when pre-incubated with the enzyme for up to 60 minutes. The competitive natures of Validamycin A and MDL 25,637 were also altered during pre-incubation with enzyme such that the compounds behaved as mixed inhibitors under these conditions. Taken together these results suggest that the inhibitory action of Validamycin A and MDL 25,637 on trehalase is of a slow-binding nature. A trehalase-specific affinity resin was synthesized by covalently coupling Validamycin A to Sepharaose 6B. This resin was used to purify D. discoideum trehalase to near homogeneity in a two-step procedure. SDS-PAGE of affinity purified trehalase, and silver staining or in situ staining for trehalase activity, revealed a major protein species of 42 kDa, exhibiting trehalase activity, and 2 minor protein species of approximately 45 kDa and 49 kDa. Since Validamycin A demonstrates strict binding specificity for trehalase, Validamycin A-Sepharose has potential and novel applications in rapid, large scale, purification of trehalases from a variety of species origins. --------------------------------------------------------------------- Inactivation of two Dictyostelium Genes, DdPIK1 and DdPIK2, encoding Proteins Related to Mammalian Phosphatidylinositide 3-kinases, results in Defects in Endocytosis, Lysosome to Post-lysosome Transport and Actin Cytoskeleton Organization Greg Buczynski, Bryon Grove1, Anson Nomura2, Maurice Kleve3, John Bush3, Richard A. Firtel2, and James Cardelli Department of Microbiology and Immunology, LSU Medical Center Shreveport, LA 71130 3Department of Biology and Altheimer Microscopy Laboratory University of Arkansas at Little Rock, Little Rock, AK 72204 2Center for Molecular Genetics, University of California at San Diego La Jolla, CA 92093-0634 J. Cell Biol., in press Phosphatidylinositide 3-kinases (PI 3-kinases) have been implicated in controlling cell proliferation, actin cytoskeleton organization and the regulation of vesicle trafficking between intracellular organelles. There are at least 3 genes in Dictyostelium discoideum, DdPIK1, DdPIK2 and DdPIK3, encoding proteins most closely related to mammalian 110 kDa PI 3-kinases in amino acid sequence within the kinase domain. A mutant disrupted in DdPIK1 and DdPIK2 (Ęddpik1/ddpik2) grows very slowly in liquid medium in shaking cultures. Using FITC-dextran (FD) as a fluid phase marker, we determined that the mutant strain was impaired in pinocytosis but normal in phagocytosis of beads or bacteria. Microscopic and biochemical approaches indicated that the transport rate of fluid-phase from acidic lysosomes to non-acidic post-lysosomal vacuoles was reduced in mutant cells resulting in a reduction in efflux of fluid phase. Mutant cells were also almost completely devoid of large post-lysosomal vacuoles as determined by transmission EM. However, Ęddpik1/ddpik2 cells functioned normally in the regulation of other membrane traffic. For instance, radio-label pulse-chase experiments indicated that the transport rates along the secretory pathway and the sorting efficiency of the lysosomal enzyme mannosidase were normal in the mutant strain. Furthermore, the contractile vacuole network of membranes (probably connected to the endosomal pathway by membrane traffic) was functionally and morphologicallyy normal in mutant cells. Light microscopy revealed that Ęddpik1/ddpik2 cells appeared smaller and more irregularly shaped than wild-type cells; 1-3% of the mutant cells were also connected by a thin cytoplasmic bridge. Scanning EM indicated that the mutant cells contained numerous filopodia projecting laterally and vertically from the cell surface, and fluorescent microscopy indicated that these filopodia were enriched in F-actin which accumulated in a more cortical pattern in control cells. Finally, Ęddpik1/ddpik2 cells responded and moved more rapidly towards cAMP. Together, these results suggest that Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the endosomal pathway, and function in the regulation of cell shape and movement perhaps through changes in actin organization. --------------------------------------------------------------------- Precocious Sporulation and Developmental Lethality in yelA Null Mutants of Dictyostelium Nir Osherov, Nancy Wang and William F. Loomis* Center for Molecular Genetics, Department of Biology,University of California San Diego, La Jolla CA 92093 Developmental Genetics, in press A novel developmental gene, yelA, has been found that plays an essential role in regulating terminal differentiation of Dictyostelium discoideum. Strains in which yelA is disrupted by plasmid insertion are arrested at the tight mound stage but accumulate the bright yellow pigment characteristic of mature sori. Although these mutant strains do not form fruiting bodies, many of the cells encapsulate within the mounds. Sporulation occurs about 6 hours earlier in yelA- cells than in wild type cells, accompanied by precocious expression of a prespore gene, spiA. However, the spores are defective and lose viability over a period of several hours. Unencapsulated cells also die unless they are dissociated from the mounds and shaken in suspension. The yelA gene was isolated by plasmid rescue and found to encode a protein of 102 kDa in which the N-terminal sequence shows significant similarity to domains found in the eIF-4G subunits of the translational initiation complex eIF-4F. In wild type cells yelA mRNA first accumulates at 8 hours of development and is maintained in both prespore and prestalk cells until culmination when it is found only is stalk cells. Mutations in yelA can partially suppress the block to sporulation in mutant strains in which either of the prestalk genes tagB or tagC is disrupted such that an encapsulation signal is not produced. It appears that premature encapsulation is normally inhibited by YelA until a signal is received from prestalk cells during culmination. --------------------------------------------------------------------- A cell-density sensing factor regulates the lifetime of a chemoattractant-induced Ga -GTP conformation Derrick T. Brazill1, Robert Gundersen2, and Richard H. Gomer1* 1Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, MS-140, Rice University, Houston, TX 77251-1892, 2Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, ME 04469-5735 FEBS letters, in press Abstract Starving Dictyostelium discoideum cells monitor the local density of other starving cells by simultaneously secreting and sensing CMF. CMF regulates signal transduction through the chemoattractant cAMP receptor, cAR1. cAR1 activates a heterotrimeric G protein by stimulating Ga2 to release GDP and bind GTP. We show here that the rate of cAMP-stimulated GTP hydrolysis in membranes from cells exposed to CMF is roughly four times slower than in membranes from untreated cells, even though the rate of GTP binding is the same. This hydrolysis is abolished in cells lacking Ga2. Our data thus suggest that CMF regulates cAMP signal transduction in part by prolonging the lifetime of the Ga2-GTP complex. --------------------------------------------------------------------- rZIP, a RING-Leucine Zipper Protein that Regulates Cell Fate Determination during Dictyostelium Development Peter Balint-Kurti, Gail Ginsburg, Octavio Rivero-Lezcano and Alan R. Kimmel MMDS, 6/B1-22, Laboratory of Cellular and Developmental Biology NIDDK , NIH Bethesda, MD 20892-2715 Development, in press Summary rZIP is an ~32 kDa, multi-domain protein of Dictyostelium discoideum whose structural motifs include a RING (zinc-binding) domain, a leucine zipper, a glutamine repeat, an SH3-binding region and a consensus phosphorylation site for MAP kinase. In vitro, rZIP forms homodimers and interacts specifically with the SH3 domain(s) of the Nck adaptor protein. rZIP is expressed maximally during cell differentiation at approximately equivalent levels in all cells. Disruption of the rZIP gene rzpA results in altered cellular aggregation, impaired slug migration, and aberrant patterning of prespore and prestalk cells, the major progenitor classes. In rzpA- strains, prespore-specific genes are overexpressed and prestalk expression zones are reduced. Conversely, constitutive overexpression of rzpA markedly decreases prespore-specific gene expression and significantly increases the expression of prestalk-specific genes. Further, induced transdifferentiation of prespore cells into prestalk cells is inhibited in rzpA- slugs. In light of these patterning defects, we suggest that the RING/zipper protein rZIP plays an important role in early cell fate decisions in Dictyostelium, acting as a positive regulator of prestalk differentiation and an inhibitor of prespore differentiation. --------------------------------------------------------------------- [End CSM News, volume 8, number 4]