Dicty News Electronic Edition Volume 15, number 6 September 16, 2000 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" ========================== Post-doctoral position ========================== A position is available in the laboratory of Richard Kessin at the Department of Anatomy and Cell Biology of The Medical Center of Columbia University in New York. Our laboratory studies the role of regulated proteolysis in the regulation of Dictyostelium development. Specifically, we study E3 and E4 components of the ubiquitin-proteolytic system. See the Website listed below for details. There is also a project that uses Dictyostelium to examine the mechanism of pathogenesis of Legionella pneumoniae, the agent of Legionaire's disease. This project is being carried out in conjunction with the laboratory of Dr. Howard Shuman, who is in The Department of Microbiology at Columbia. The laboratory is part of a major research center and is in a well equipped department. Experience in molecular biology and biochemistry is important. Salary is commensurate with experience. The university provides assistance with visas and housing. Contact Richard Kessin (rhk2@Columbia.edu). For more information, see our Website at: http://cpmcnet.columbia.edu/dept/gsas/anatomy/Faculty/Kessin/index.html ============== Abstracts ============== Mapping the functional surface of domain 2 in the gelsolin superfamily. 1Puius YA, 1Fedorov EV, 2Eichinger L, 2Schleicher M, 1Almo SC 1Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA. 2 ABI/Cell Biology, Ludwig-Maximilians-University Muenchen, Schillerstr. 42; 80336 Muenchen, Germany. Biochemistry 2000 May 9;39(18):5322-31 The crystal structure of the F-actin binding domain 2 of severin, the gelsolin homologue from Dictyostelium discoideum, has been determined by multiple isomorphous replacement and refined to 1.75 A resolution. The structure reveals an alpha-helix-beta-sheet sandwich similar to the domains of gelsolin and villin, and contains two cation-binding sites, as observed in other domain 1 and domain 2 homologues. Comparison of the structures of several gelsolin family domains has identified residues that may mediate F-actin binding in gelsolin domain 2 homologues. To assess the involvement of these residues in F-actin binding, three mutants of human gelsolin domain 2 were assayed for F-actin binding activity and thermodynamic stability. Two of the mutants, RRV168AAA and RLK210AAA, demonstrated a lowered affinity for F-actin, indicating a role for those residues in filament binding. Using both structural and biochemical data, we have constructed a model of the gelsolin domain 1-domain 2-F-actin complex. This model highlights a number of interactions that may serve as positive and negative determinants of filament end- and side-binding. ---------------------------------------------------------------------------- A syntaxin 7 homologue is present in Dictyostelium discoideum endosomes and controls their homotypic fusion Aleksandra Bogdanovic1, Franz Bruckert1, Takahiro Morio2 and Michel Satre1 1Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Département de Biologie Moléculaire et Structurale, 38054 Grenoble Cedex 9, France 2Institute of Biological Sciences, University of Tsukuba, Tsukuba, Japan to be published in the Journal of Biological Chemistry Summary Endo-phagocytic activity is prominent in Dictyostelium discoideum and makes it a good model organism to study the molecular organization of membrane traffic in this pathway. We have identified a syntaxin 7 homologue (26% identity and 54% similarity to human syntaxin 7) in Dictyostelium cDNA and genomic databanks. In addition to the Habc and H3 helices and the C-terminal transmembrane domain characteristic of syntaxins, this protein contains a repetitive N-terminal extension of 68 amino acids. We first showed that Dictyostelium syntaxin 7 was able to form a complex with NSF,alpha-SNAP and gamma-SNAP. Its intracellular localization was then studied by cell fractionation techniques and magnetic purification of the endocytic compartments. Most of D. discoideum syntaxin 7 is contained in endosomes. Finally, an in vitro endosome homotypic fusion assay (Laurent et al. 1998, J. Biol. Chem. 273: 793-799) was used to study a possible role for syntaxin 7 in this process. Purified anti-syntaxin 7 antibodies and a recombinant soluble fragment of syntaxin 7 both strongly inhibited fusion activity, indicating that this protein was necessary for endosome-endosome fusion. These results demonstrate the importance of this syntaxin 7 homologue in the early phases of Dictyostelium endo-phagocytic pathway. ---------------------------------------------------------------------------- Glycogen synthase kinase-3 (GSK-3) enhances nuclear export of a Dictyostelium STAT protein Rebecca S. Ginger1,3 *, Emma C. Dalton2*, W. Jonathan Ryves2, Masashi Fukuzawa1 Jeffrey G. Williams1 and Adrian J. Harwood2,4. 1. Department of Anatomy and Physiology, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK. 2. MRC, Laboratory for Molecular Cell Biology, UCL (University College London), Gower St, London WC1E 6BT, UK. 020 7679 7257 3. Present Address: Unilever Research, Colworth House, Sharnbrook, Beds MK44 1LQ 4. To whom correspondence should be addressed. e-mail: a.harwood@ucl.ac.uk * Both authors contributed equally to this work EMBO, in press ABSTRACT Extracellular cAMP stimulates the rapid tyrosine phosphorylation and nuclear translocation of the Dictyostelium STAT protein Dd-STATa. Here, we show that it also induces serine phosphorylation by GskA, a homologue of GSK-3. Tyrosine phosphorylation occurs within 10 seconds of stimulation, whereas serine phosphorylation takes 5 minutes, matching the kinetics observed for the cAMP regulation of GskA. Phosphorylation by GskA enhances nuclear export of Dd-STATa. The phosphorylated region, however, is not itself a nuclear export signal (NES) and we identify a region elsewhere in the protein that mediates nuclear export. These results suggest a biphasic regulation of Dd-STATa, in which extracellular cAMP initially directs nuclear import and then, via GskA, promotes its subsequent export. It also raises the possibility of an analogous regulation of STAT nuclear export in higher eukaryotes. ---------------------------------------------------------------------------- [End Dicty News, volume 15, number 6]