Dicty News Electronic Edition Volume 13, number 4 August 7, 1999 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 ============= Assessing the role of ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization Angelika A. Noegel(1), Francisco Rivero(1), Richard Albrecht(2), Klaus-Peter Janssen(4), Jana Köhler(2), Carole A. Parent(3) and Michael Schleicher(4)* (1)Institut für Biochemie I, Medizinische Einrichtungen der Universität zu Köln, Joseph-Stelzmann-Str. 52, 50931 Köln, (2) Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, (3) Department of Biological Chemistry, Johns Hopkins University, School of Medicine, North Wolfe St., Baltimore, Maryland 21205, USA, (4) Institut für Zellbiologie der Ludwig-Maximilians-Universität München, Schillerstrasse 42, 80336 München, F.R.G. J. Cell Sci., in press CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP2) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas presence of the N-terminal domain seems to be required for PIP2-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected. ---------------------------------------------------------------------------- A Dictyostelium protein binds to distinct oligo(dA).oligo(dT) DNA sequences in the C-module of the retrotransposable element DRE Jens Horn (1), Anja Dietz-Schmidt (1), Ilse Z|ndorf (1), Jirtme Garin (2), Theodor Dingermann (1) and Thomas Winckler (2) (1) Institut f|r Pharmazeutische Biologie, Universitaet Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany and (2) Laboratoire de Chimie des Protiines, DBMS/CP, CEN-G Grenoble Cedex 09, France Eur. J. Biochem., in press The genome of the eukaryotic microbe Dictyostelium discoideum contains some 200 copies of the non-long terminal repeat retrotransposon DRE. Among several unique features of this retroelement, DRE is transcribed in both directions leading to the formation of partially overlapping plus strand and minus strand RNAs. The synthesis of minus strand RNAs is controlled by the C-module, a 134 bp DNA sequence located at the 3' end of DRE. A nuclear protein (CMBF) binds to the C-module via interaction with two nearly homopolymeric 24 bp oligo(dA).oligo(dT) sequences. The DNA-binding drugs distamycin and netropsin, which bind to A.T-rich DNA sequences in the minor groove, efficiently competed for the binding of CMBF to the C-module. The CMBF-encoding gene, cbfA, was isolated and a DNA-binding domain was mapped to a 25 kDa carboxy-terminal region of the protein. A peptide motif involved in the binding of A.T-rich DNA by high mobility group-I proteins ("GRP" box) was identified in the deduced CMBF protein sequence, and exchange of a consensus arginine residue for alanine within the CMBF GRP box abolished the interaction of CMBF with the C-module. The current data support that CMBF binds to the C-module by detecting its long-range DNA conformation and interacting with A.T base pairs in the minor groove of oligo(dA).oligo(dT) stretches. ---------------------------------------------------------------------------- The multigene immunophilin family of Dictyostelium discoideum. Characterisation of microsomal and mitochondrial cyclophilin isoforms Aurelie Tapparo1, Sylvie Kieffer2, Francois Cretin3, Michel Satre1 and Gerard Klein1 1Lab. Biochemistry and Biophysics of Integrated Systems, (UMR314 CNRS), 2Lab. Protein Chemistryand 3Lab. Immunochemistry (U238 INSERM), Departement of Molecular and Structural Biology, CEA-Grenoble, France. Biochimie, in press. SUMMARY The sequences of cypB and cypD genes encoding Dictyostelium discoideum cyclophilin (Cyp) isoforms have been determined. cypB is expressed as a 197 amino acid protein, which contains a 22 amino acid-long signal sequence, characteristic of endoplasmic reticulum localisation signals, and that is cleaved in the mature protein. Mature CypB has a molecular mass of 18 986 Da. The cypD gene encodes a 174 amino acid protein with a predicted molecular mass of 19 016 Da. Its sequence reveals no targeting sequence. From the mass spectrometry analysis of affinity-purified cyclophilins from different subcellular compartments, we localised the previously described CypA (Barisic, K., Mollner, S., Noegel, A. A., Gerisch, G. & Segall, J. E. (1991). Developmental Genetics 12, 50-53) in cytosol, CypB in microsomes and CypD in mitochondria, respectively. CypA, -B and -D are inhibited by cyclosporin A, despite a W to H substitution in the cyclosporin A binding domain of CypD. The expression of cypA mRNA is constant during differentiation, whereas the mRNA level of both cypB and cypD is regulated and decreases steadily during the 24 hours of development. Besides the three cyclophilin genes, D. discoideum also harbours several genes coding for FK506-binding protein (FKBP) homologs. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 4]