Dicty News Electronic Edition Volume 10, number 9 March 28, 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 =========== LOCALISATION OF GLYCOPROTEINS CONTAINING TYPE 3 O-LINKED GLYCOSYLATION TO MULTILAMELLAR BODIES IN DICTYOSTELIUM DISCOIDEUM. Emslie, K.R., Birch, D., Champion, A.C. and Williams, K.L. * Department of Biological Sciences, Macquarie University Sydney NSW 2109 Int. J. Parasitology ABSTRACT Glycosylation of proteins in Dictyostelium discoideum has been recently classified into several types based on structural and mutational studies. Type 3 O-linked glycosylation contains fucose and N-acetyl-glucosamine and is recognised by the carbohydrate-specific monoclonal antibody MUD 62. The developmentally regulated cysteine proteinase, ddCP38B, is the major protein recognised by both MUD 62 and a second carbohydrate-specific monoclonal antibody, MUD 166, in vegetative cells using bacteria as a food source. Using immunofluorescence and immunogold labelling, glycoproteins carrying the MUD 62 and MUD 166-specific epitopes have been localised intracellularly to the lysosomal network and multilamellar bodies of D. discoideum. These bodies contain remnants of undigested bacteria and are egested from the cell prior to starvation-induced aggregation. These results suggest that extracellular glycoproteins carrying the Type 3 O-linkage, such as ddCP38B, may not be secreted via the conventional pathway but may be released into the medium following targeting to the multilamellar bodies. ------------------------------------------------------------------------- A Distinct 14-Residue Site Triggers Coiled-Coil Formation in Cortexillin I Michel O. Steinmetz1, Alexander Stock2, Therese Schulthess3, Ruth Landwehr3, Ariel Lustig3, Jan Faix2, Guenther Gerisch2, Ueli Aebi1, and Richard A. Kammerer3 1M.E. Mueller Institute for Microscopy, Biozentrum, University of Basel, CH-4056 Basel, Switzerland, 2Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany, 3Department of Biophysical Chemistry, Biozentrum, University of Basel, CH-4056 Basel, Switzerland EMBO J. in press. Abstract: We have investigated the assembly process of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed two-stranded parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation, and electron microscopy. Deletion mapping identified a distinct 14-residue site, Arg311 to Asp324, within the Ir coiled coil which was absolutely necessary for dimer formation, indicating that heptad repeats, by themselves, are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four-helix, rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16-residue long peptide containing the distinct coiled-coil "trigger" site Arg311 to Asp324 yielded ~30% helix formation, as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311 to Asp324 behaves as an autonomous helical folding unit, and they further suggest that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I. ------------------------------------------------------------------------- Rules for the addition of O-linked N-acetylglucosamine to secreted proteins in Dictyostelium discoideum: In vivo studies on glycosylation of mucin MUC1 and MUC2 repeats Eva JUNG, Andrew A. GOOLEY, Nicolle H. PACKER, Peter KARUSO and Keith L. WILLIAMS MUCAB (Macquarie University Centre for Analytical Biotechnology), School of Biological Sciences and School of Chemistry, Macquarie University, Sydney, NSW 2109, Australia Eur. J. Biochem., in press One class of O-glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N-acetylglucosamine to serine and threonine residues on secreted or membrane bound proteins at an early stage of development. A previously developed in vivo approach for the identification of acceptor sites for O-glycosylation was used to further characterise the specificity of the UDP-GlcNAc: polypeptide N-acetylglucosaminyltransferase(s). Glutathione-S-transferase fusion proteins were constructed to express and secrete the mucin peptide repeat for MUC1 (PDT1RPAPGS1T2APPAHGVT3S2A) and a MUC2-like peptide (PT1T2T3PIT4T5T6T7T8T9VT10PT11PT12PT13GT14QT15), respectively. Monosaccharide analysis, electrospray-ionisation mass spectrometry and protein sequencing showed that the modification is a single N-acetylglucosamine attached to certain threonine residues. The MUC1 repeat was glycosylated on T2 and T3 and there were no modifications on T1 or on S1 and S2. The MUC2 glycopeptide was glycosylated on T1, T3, T5, T7, T9, T10, T11, T12, T13 and T14. Our results show that the D. discoideum glycosylation apparatus incorporates GlcNAc residues into peptide sequences similar to those reported for the addition of GalNAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in alpha configuration as determined by NMR studies. ------------------------------------------------------------------------- Spalten, a protein containing Galpha-protein-like and PP2C domains, is essential for cell-type differentiation in Dictyostelium Laurence Aubry and Richard A. Firtel Genes and Deveopment, in press ABSTRACT We have identified a novel gene, Spalten (Spn) that is essential for Dictyostelium multicellular development. Spn encodes a protein with an N-terminal domain that shows very high homology to Galpha- protein subunits, a highly charged inter-region, and a C-terminal domain that encodes a functional PP2C. Spn is essential for development past the mound stage, being required cell autonomously for prestalk gene expression and non-autonomously for prespore cell differentiation. Mutational analysis demonstrates that the PP2C domain is the effector domain and is essential for Spn function, while the Galpha-like domain is required for membrane targeting and regulation of Spn function. Moreover, Spn carrying mutations in the Galpha-like domain that do not affect membrane targeting but affect specificity of guanine nucleotide binding in known GTP-binding proteins are unable to fully complement the spn- phenotype, suggesting that the Galpha-like domain regulates Spn function either directly or indirectly by mediating its interactions with other proteins. Our results suggest that Spn encodes a signaling molecule with a potentially novel Galpha-like regulatory domain. ------------------------------------------------------------------------- [End Dicty News, volume 10, number 9]