Dicty News Electronic Edition Volume 10, number 14 June 6, 1998 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== A rapid, small scale method for characterization of plasmid insertions in the Dictyostelium genome C. BARTH, D.J. FRASER AND P.R. FISHER School of Microbiology, La Trobe University, Bundoora 3083, Melbourne, Australia Nucleic Acids Research. In Press. Summary A rapid, simple method for characterization of plasmid insertions in the Dictyostelium discoideum genome was developed. It is based on the capability of linear plasmid multimers in the insertions to recircularize efficiently in Escherichia coli cells. This recombinational recircularization of plasmid multimers provides a highly sensitive and reliable tool for determining whether individual Dictyostelium transformants resulted from restriction enzyme-mediated integration (REMI) or from recombinational integration of plasmid (RIP). The method also reveals any rearrangements in RIP insertions and provides an estimate of the vector copy number in any particular transformant. ------------------------------------------------------------------------- Two Proteins of the Dictyostelium Spore Coat Bind to Cellulose in vitro Yunyan Zhang, Ross D. Brown, Jr., and Christopher M. West Department of Anatomy & Cell Biology, University of Florida College of Medicine, and Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, Gainesville, FL 32610 USA Biochemistry, in press Summary: The spore coat of Dictyostelium contains nine different proteins and cellulose. Interactions between protein and cellulose were investigated using an in vitro binding assay. Proteins extracted from coats with urea and 2-mercaptoethanol could, after removal of urea by gel filtration, efficiently bind to particles of cellulose (Avicel), but not Sephadex or Sepharose. Two proteins, SP85 and SP35, were enriched in the reconstitution, and retained their cellulose binding activities after purification by ion exchange chromatography under denaturing conditions to suppress protein-protein interactions. Neither protein exhibited cellulase activity, though under certain conditions SP85 copurified with a cellulase activity which appeared after germination. Amino acid sequencing indicated that SP85 and SP35 are encoded by the previously described pspB and psvA genes. This was confirmed for SP85 by showing that natural Mr polymorphisms correlated with changes in the number of tetrapeptide-encoding sequence repeats in pspB. Using PCR to reconstruct missing elements from the recombinogenic middle region of pspB, SP85 was shown to consist of three sequence domains separated by two groups of the tetrapeptide repeats. Expression of partial pspB cDNAs in E. coli showed that cellulose- binding activity resided in the Cys-rich COOH-terminal domain of SP85. This cellulose-binding activity can explain SP85’s ultrastructural colocalization with cellulose in vivo. Amino acid composition and antibody binding data showed that SP35 is derived from the Cys-rich N-terminal region of the previously described psvA protein. SP85 and SP35 may link other proteins to cellulose during coat assembly and germination. ----------------------------------------------------------------------------------- The Cytoplasmic F-box Binding Protein SKP1 Contains a Novel Pentasaccharide Linked to Hydroxyproline in Dictyostelium Patana Teng-umnuay, Howard R. Morris, Anne Dell, Maria Panico, Thanai Paxton, and Christopher M. West Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida 32610-0235, USA; Department of Biochemistry, Imperial College, London SW7 2AY, UK J. Biol. Chem., in press. Summary: SKP1 is involved in the ubiquitination of certain cell cycle and nutritional regulatory proteins for rapid turnover. SKP1 from Dictyostelium has been known to be modified by an oligosaccharide containing Fuc and Gal, which is unusual for a cytoplasmic or nuclear protein. To establish how it is glycosylated, SKP1 labeled with [3H]Fuc was purified to homogeneity and digested with endo-Lys-C. A single radioactive peptide was found after 2-dimensional HPLC. Analysis in a Q-TOF mass spectrometer revealed a predominant ion with a novel mass. MS/MS analysis yielded a set of daughter ions which identified the peptide and showed that it was modified at Pro143. A second series of daughter ions showed that Pro143 was hydroxylated and derivatized with a potentially linear pentasaccharide, Hex-Hex-Fuc- Hex-HexNAc-(HyPro). The attachment site was confirmed by Edman degradation. GC-MS analysis of TMS-derivatives of overexpressed SKP1 after methanolysis showed the HexNAc to be GlcNAc. Exoglycosidase digestions of the glycopeptide from normal SKP1 and from a fucosylation mutant, followed by MALDI-TOF-MS analysis, showed that the sugar chain consisted of D-Galpalpha1,6-D-Galpalpha1,L-Fucpalpha1,2-D- Galpbeta1,3GlcNAc. MALDI-TOF-MS analysis of all SKP1 peptides resolved by RP-HPLC showed that SKP1 was only partially hydroxylated at Pro143, and that all hydroxylated SKP1 was completely glycosylated. Thus SKP1 is variably modified by an unusual linear pentasaccharide, suggesting the localization of a novel glycosylation pathway in the cytoplasm. ------------------------------------------------------------------------- G-protein beta-subunit null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton Barbara Peracino, Jane Borleis*, Tian Jin*, Monika Westphal#, Jean-Marc Schwartz#, Lijun Wu°, Enrico Bracco, Günther Gerisch#, Peter Devreotes* and Salvatore BozzaroÝ J. Cell Biology, in press ABSTRACT Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amoebae. Deletion of the unique G-protein beta-subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis or random motility. Constitutive expression of wild-type beta-subunit restored phagocytosis and normal development. Chemoattractants, released by cells or bacteria, trigger typical transient actin polymerization responses in wild-type cells. In beta-subunit null cells, and in a series of beta subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of GFP-actin transfected cells showed that beta-subunit null cells were defective in re-shaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signalling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phopholipase C and intracellular Ca2+ mobilization inhibit phagocytosis, suggesting the possible involvement of these effectors in the process. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 14]