Dicty News Electronic Edition Volume 10, number 8 March 21, 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 =========== UGUS, A REPORTER FOR USE WITH DESTABILIZING N-TERMINI. Davide Zaccaria, Roberta Greco, Harry MacWilliams§, Salvatore Bozzaro, and Adriano Ceccarelli*. Dipartimento di Scienze Cliniche e Biologiche - Universita' di Torino, Ospedale S.Luigi, 10043 Orbassano (Italy), and § Zoologisches Institut, Ludwig-Maximilians-Universitat, Luisenstrasse 14, 80333 Muenchen (Germany) *To whom correspondence should be sent ABSTRACT Due to constraints in vector construction, reporter polypeptides often carry N-terminal sequences of extraneous origin. Since protein half-life can be influenced by small determinants in the N-terminus, such foreign sequences can destabilize proteins and compromise results of reporter- based studies. We provide a real-life example of this problem (destabilizing sequences derived from a ribosomal protein) and show that it can be solved with the ubiquitin fusion technique, in which ubiquitin sequences are placed upstream of the reporter, in our case beta-glucuronidase. Post-translational processing by characterized pathways removes the ubiquitin together with destabilizing sequences, generating a stable reporter whose N-terminus is constant. ------------------------------------------------------------------------- An intersection of the cAMP/PKA and two-component signal transduction systems in Dictyostelium Peter A. Thomason1, David Traynor1, Guy Cavet* Wen-Tsan Chang2, Adrian J. Harwood* and Robert R. Kay MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England EMBO J in press Summary: Terminal differentiation of both stalk and spore cells in Dictyostelium can be triggered by activation of cAMP-dependent protein kinase (PKA). A screen for mutants where stalk and spore cells mature in isolation produced three genes which may act as negative regulators of PKA: rdeC (encoding the PKA regulatory subunit), regA and rdeA. The biochemical properties of RegA were studied in detail. One domain is a cAMP phosphodiesterase (Km ~5 micro-Molar). The other domain is homologous to response regulators of two-component signal transduction systems. It can accept phosphate from acetyl phosphate in a reaction typical of response regulators, with transfer dependent on Asp212, the predicted phospho-acceptor. RegA phosphodiesterase activity is stimulated up to 8-fold by the phospho-donor phosphoramidate, with stimulation again dependent on Asp212. This indicates that phosphorylation of the response regulator domain activates the phosphodiesterase domain. Over-expression of the response regulator domain in wild-type cells phenocopies a regA null. We interpret this dominant negative effect as due to a diversion of the normal flow of phosphates from RegA, thus preventing its activation. Mutation of rdeA is known to produce elevated cAMP levels. We propose that cAMP breakdown is controlled by a phospho-relay system which activates RegA, and may include RdeA. Cell maturation should be triggered when this system is inhibited. ------------------------------------------------------------------------- Evidence that the RDEA protein is a component of a multistep phosphorelay modulating rate of development in Dictyostelium Wen-Tsan Chang, Peter A. Thomason*, Julian D. Gross and Peter C. Newell Department of Biochemistry, University of Oxford, Oxford OX1 3QU, U.K. and (*) MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, U.K. EMBO J. in press. We have isolated an insertional mutant of Dictyostelium discoideum that aggregated rapidly and formed spores and stalk cells within 14 hours of development instead of the normal 24 hours. We have shown by parasexual genetics that the insertion is in the rdeA locus and have cloned the gene. It encodes a predicted 28-kD protein (RDEA) that is enriched in charged residues and is very hydrophilic. Constructs with the DNA for the C-myc epitope or for the green fluorescent protein indicate that RDEA is not compartmentalised. RDEA displays homology around a histidine residue at amino acid 65 with members of the H2-module family of phosphotransferases that participate in multistep phosphoryl relays. Replacement of this histidine rendered the protein inactive. The mutant is complemented by transformation with the YPD1 gene of S. cerevisiae, itself an H2-module protein. We propose that RDEA is part of a multistep phosphorelay system that modulates the rate of development. ------------------------------------------------------------------------- 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 ------------------------------------------------------------------------- [End Dicty News, volume 10, number 8]