Dicty News Electronic Edition Volume 19, number 8 October 12, 2002 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============================== Postdoc Position Available ============================== Postdoctoral position in the Wellcome Trust Biocentre at the University of Dundee A postdoctoral position is available in Dr. Inke Nthke s laboratory to join a team studying the function of the Adenomatous Polyposis Coli tumour suppressor protein (APC). The project will involve using Dictyostelium Discoideum to investigate the role of APC in cytoskeletal dynamics and organisation. The general aim of work in the laboratory is to determine how the diverse functions of APC relate to its role in cancer and how these functions are co-ordinated and regulated. Experimental approaches include general cell- and molecular biology techniques combined with high-resolution fluorescence microscopy and use of Dictyostelium as a model organism. The candidate will join a small, dynamic, highly interactive research team with active collaborations worldwide. A proven research ability and motivation are required and experience in working with Dictyostelium is requisite. Experience in general cell biology and molecular biology is essential. The position is available immediately for 2 years in the first instance. For informal enquiries, please contact Dr. Inke S. Nthke (+ 44 1382 345821; e-mail: i.s.nathke@dundee.ac.uk). Applications should be directed to: Janette Cordiner, School Administrator, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH; j.m.cordiner@dundee.ac.uk. Please cite reference SC/763/1 on the application. The Wellcome Trust Biocentre is part of the internationally renowned WTB/MSI complex at the University of Dundee. The complex provides an extremely interactive research environment and houses nearly 400 research staff. Further information about research in the WTB/MSI complex can be obtained at http://www.dundee.ac.uk/biocentre/. ============= Abstracts ============= CaMBOT: Profiling and Characterizing Calmodulin Binding Proteins Danton H. O Day Department of Zoology, University of Toronto at Mississauga, Mississauga, ON L5L 1C6 CANADA Cellular Signalling (in press) Abstract Calmodulin (CaM) is an essential calcium-binding protein that binds to and activates a diverse population of downstream targets (calmodulin binding proteins; CaMBPs) that carry out its critical signalling functions. In spite of the central importance of CaM in Ca2+-mediated signal transduction pathways in all eukaryotes, most CaMBPs remain to be identified and characterized. SDS-PAGE followed by gel overlay with recombinant, metabolically radiolabelled CaM (Calmodulin Binding Overlay Technique, CaMBOT) is a valuable method for following behavioral, developmental, forensic and physiological changes in total CaMBP populations and to identify candidate CaMBPs for further study. CaMBOT has also been adapted to isolate cDNAs encoding novel CaMBPs in various organisms. Recently the method was used to examine the CaMBP complement encoded by the Arabidopsis genome and to identify a new family of transcription activators. To add to its diversity, CaMBOT may be useful for finding target proteins for work on phytoremediation and for the screening of pharmaceuticals and toxic agents that, directly or indirectly, affect CaM and its target proteins. This review discusses all of these topics and the role of CaMBOT in characterizing a functional unit of the proteome proteins regulated by calmodulin. ----------------------------------------------------------------------------- Myosin Heavy Chain Kinase B participates in the regulation of myosin assembly into the cytoskeleton. Maribel Rico and Thomas T. Egelhoff Department of Physiology and Biophysics, Case Western Reserve University, Cleveland. OH. 44106-4970 Journal of Cellular Biochemistry, in press Abstract Myosin II plays critical roles in events such as cytokinesis, chemotactic migration, and morphological changes during multicellular development. The amoeba Dictyostelium discoideum provides a simple system for the study of this contractile protein. In this system, myosin II filament assembly is regulated by myosin heavy chain (MHC) phosphorylation in the tail region of the molecule. Earlier studies identified an alpha- kinase, myosin heavy chain kinase A (MHCK A), which phosphorylates three mapped threonine residues in the myosin tail, driving myosin disassembly. Using molecular and genomic approaches we have identified a series of related kinases in Dictyostelium. The enzyme MHCK B shares with MHCK A a domain organization that includes a highly novel catalytic domain coupled to a carboxyl-terminal WD repeat domain. We have engineered, expressed and purified a FLAG-tagged version of the novel kinase. In the present study, we report detailed biochemical and cellular studies documenting that MHCK B plays a physiological role in the control of Dictyostelium myosin II assembly and disassembly during the vegetative life of Dictyostelium amoebae. The presented data supports a model of multiple related MHCKs in this system, with different regulatory mechanisms and pathways controlling each enzyme. ----------------------------------------------------------------------------- Dictyostelium discoideum has a single diacylglycerol kinase gene with similarity to mammalian theta isoforms Marc A. de la Roche1, Janet L. Smith2, Maribel Rico3, Silvia Carrasco4, Isabel Merida4, Lucila Licate3, Graham P. Ct1, and Thomas T. Egelhoff3# 1. Department of Biochemistry Queen s University Kingston, Ontario K7L 3N6, Canada 2. Boston Biomedical Research Institute 64 Grove St. Watertown, MA 02472-2829 3. Department of Physiology and Biophysics Case Western Reserve School of Medicine Cleveland, OH 44016-4970 4 Department of Immunology and Oncology National Center for Biotechnology Campus de Cantoblanco Madrid 28049, Spain # Corresponding author Biochemical Journal (online as preprint), in press Synopsis Diacylglycerol kinases (DGKs) phosphorylate the neutral lipid diacylglycerol (DG) to produce phosphatidic acid (PA). In mammalian systems DGKs are a complex family of at least 9 isoforms that are thought to participate in downregulation of DG-based signaling pathways and perhaps activation of PA-stimulated signaling events. We report here that the simple protozoan amoeba Dictyostelium discoideum appears to contain a single gene encoding a DGK enzyme. This gene, dgkA, encodes a deduced protein that contains three C1-type cysteine-rich repeats, a DGK catalytic domain most closely related to the theta subtype of mammalian DGKs, and a carboxyl-terminal segment containing a proline/glutamine-rich region and a large aspargine-repeat region. This gene corresponds to a previously reported myosin II heavy chain kinase designated "MHC-PKC", but our analysis clearly demonstrate that this protein does not, as suggested by earlier data, contain a protein kinase catalytic domain. A FLAG-tagged version of DgkA expressed in Dictyostelium displayed robust diacylglycerol kinase activity. Earlier studies indicating that disruption of this locus alters myosin II assembly levels in Dictyostelium raise the intriguing possibility that DG and/or PA metabolism may play a role in controlling myosin II assembly in this system. ----------------------------------------------------------------------------- A Bifunctional Di-Glycosyltransferase Forms the Fuca1,2Galb1,3-Disaccharide on Skp1 in the Cytoplasm of Dictyostelium Hanke van der Wel, Suzanne Z. Fisher, and Christopher M. West Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235 USA J. Biol. Chem., in press Skp1 is a subunit of the SCF-family of E3-ubiquitin ligases and of other regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by a pentasaccharide with the type I blood group H antigen (Fuca1,2Galb1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85, a 768-amino acid protein whose fucosyltransferase activity maps to the C-terminal half of the protein. A strain whose FT85 gene is interrupted by a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1, suggesting that FT85 may also have b-galactosyltransferase activity. In support of this model, highly-purified native and recombinant FT85 are each able to galactosylate Skp1 from FT85-mutant cells. Site-directed mutagenesis of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1 b-galactosyltransferase activity with minimal effects on the fucosyltransferase. In addition, a recombinant form of the N-terminal region exhibits b-galactosyltransferase but not fucosyltransferase activity. Kinetic analysis of FT85 suggests that its two glycosyltransferase activities normally modify Skp1 processively but can have partial function individually. In conclusion, FT85 is a bifunctional di-glycosyltransferase that appears to be designed to efficiently extend the Skp1 glycan in vivo. ----------------------------------------------------------------------------- Evolutionary and functional implications of the complex glycosylation of Skp1, a cytoplasmic/nuclear glycoprotein associated with polyubiquitination Christopher M. West Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235 (USA), Fax +1 352 392 3305, e-mail: westcm@ufl.edu Cell and Molecular Life Sciences, in press. Protein degradation is regulatory for the cell cycle, signal transduction and gene transcription. A critical step is the selective marking of the target protein, resulting in polyubiquitination by one of a large number of E3-ubiquitin ligases. Both target marking and E3-ubiquitin ligase activity are associated with common as well as unusual posttranslational modifications. For example, hydroxylation of Pro-residues and modification of Asn-residues by high-mannose sugar chains can target the modified proteins for rapid polyubiquitination in the mammalian cytoplasm. Both prolyl hydroxylation and glycosylation also occur on Skp1, a subunit of the SCF-class of E3-ubiquitin ligases, from Dictyostelium. In this case, a pentasaccharide containing Gal, Fuc and GlcNAc is attached to the HyPro-residue. The sugars are added sequentially by enzymes that reside in the cytoplasm rather than the secretory pathway. Two of the glycosyltransferases appear to be positioned in ancient evolutionary lineages that bridge prokaryotes and eukaryotes. The first, which attaches GlcNAc to HyPro, is related to enzymes that form a-GalNAc- and a-GlcNAc-Ser/Thr linkages in the Golgi. GlcNAc is extended by a bifunctional glycosyltransferase that mediates the ordered addition of b1,3-linked Gal and a1,2-linked Fuc, using an architecture resembling that of 2-domain prokaryotic glycosyltransferases involved in glycosaminoglycan synthesis. Mutational and pharmacological perturbation of glycosylation alters the subcellular localization of Skp1 and growth properties of cells. Prolyl hydroxylation and complex O-glycosylation provide the cell with new options for epigenetic regulation of protein turnover in its cytoplasmic and nuclear compartments. ----------------------------------------------------------------------------- Molecular Cloning and Expression of a UDP-GlcNAc:Hydroxyproline Polypeptide GlcNAc-Transferase that Modifies Skp1 in the Cytoplasm of Dictyostelium Hanke van der Wela, Howard R. Morrisb,c, Maria Panicob, Thanai Paxtonb, Anne Dellb, Lee Kaplana, and Christopher M. Westa,d aDepartment of Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, FL USA 32610-0235; bDepartment of Biological Sciences, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK; cM-SCAN Research and Training Center, Silwood Park, Ascot SL5 7PZ, UK J. Biol. Chem., in press. Skp1 is a ubiquitous eukaryotic protein found in several cytoplasmic and nuclear protein complexes, including the SCF-type E3 ubiquitin ligase. In Dictyostelium, Skp1 is hydroxylated at proline-143 which is then modified by a pentasaccharide chain. The enzyme activity that attaches the first sugar, GlcNAc, was previously shown to copurify with the GnT51 polypeptide whose gene has now been cloned using a proteomics approach based on a Q-TOF hybrid mass spectrometer. When expressed in E. coli, recombinant GnT51 exhibits UDP-GlcNAc:hydroxyproline Skp1 GlcNAc-Transferase activity. Based on amino acid sequence alignments, GnT51 defines a new family of microbial polypeptide glycosyltransferases that appear to be distantly related to the catalytic domain of mucin-type UDP-GalNAc:Ser/Thr polypeptide a-GalNAc-Transferases expressed in the Golgi compartment of animal cells. This relationship is supported by the effects of site-directed mutagenesis of amino acids associated with GnT51's predicted DxD-like motif, DAH. In contrast, GnT51 lacks the NH2-terminal signal anchor sequence present in the Golgi enzymes, consistent with the cytoplasmic localization of the Skp1 acceptor substrate and the biochemical properties of the enzyme. The first glycosylation step of Dictyostelium Skp1 is concluded to be mechanistically similar to that of animal mucin type O-linked glycosylation except that it occurs in the cytoplasm rather than the Golgi compartment of the cell. ----------------------------------------------------------------------------- GenePath: a System for Automated Construction of Genetic Networks from Mutant Data Blaz Zupan, Janez Demsar, Ivan Bratko, Peter Juvan, John A. Halter, Adam Kuspa and Gad Shaulsky University of Ljubljana, Faculty of Computer and Information Science and Jozef Stefan Institute, Ljubljana, Slovenia Departments of PM&R and Division of Neuroscience, Biochemistry and Molecular Biology, Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA Bioinformatics, in press ABSTRACT Motivation: Genetic networks are often used in the analysis of biological phenomena. In classical genetics, they are constructed manually from experimental data on mutants. The field lacks formalism to guide such analysis, and accounting for all the data becomes complicated when large amounts of data are considered. Results: We have developed GenePath, an intelligent assistant that automates the analysis of genetic data. GenePath employs expert-defined patterns to uncover gene relations from the data, and uses these relations as constraints in the search for a plausible genetic network. GenePath formalizes genetic data analysis, facilitates the consideration of all the available data in a consistent manner, and the examination of the large number of possible consequences of planned experiments. It also provides an explanation mechanism that traces every finding to the pertinent data. Availability: GenePath can be accessed at http://genepath.org. Contact: gadi@bcm.tmc.edu Supplementary information: Supplementary material is available at http://genepath.org/bi-supp. ----------------------------------------------------------------------------- Tail chimeras of Dictyostelium myosin II support cytokinesis and other myosin II activities but not full development Shi Shu1, Xiong Liu1, Carole A. Parent 2, Taro Q. P. Uyeda3, and Edward D. Korn*1 1 Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, and 2 Laboratory of Cellular and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 and 3Gene Function Research Center, Tsukuba Central #4, National Institute of Advanced Industrial Science and Technology (AIST), Higashi 1-1-1, Tsukuba, Ibaraki 305-8562, Japan *Author for correspondence (e-mail: edk@nih.gov) Short title: In vivo activities of myosin II tail chimeras Key words: myosin II; cytokinesis; chemotaxis; development; Con A capping. Journal of Cell Science, in press SUMMARY Dictyostelium lacking myosin II cannot grow in suspension culture, develop beyond the mound stage or cap concanavalin A receptors and chemotaxis is impaired. Recently, we showed that the actin-activated MgATPase activity of myosin chimeras in which the tail domain of Dictyostelium myosin II heavy chain is replaced by the tail domain of either Acanthamoeba or chicken smooth muscle myosin II is unregulated and about 20 times higher than wild-type myosin. The Acanthamoeba chimera forms short bipolar filaments similar to, but shorter than, filaments of Dictyostelium myosin, and the smooth muscle chimera forms much larger side-polar filaments. We now find that the Acanthamoeba chimera expressed in myosin null cells localizes to the periphery of vegetative amoeba similarly to wild-type myosin but the smooth muscle chimera is heavily concentrated in a single cortical patch. Despite their different tail sequences and filament structures and different localization of the smooth muscle chimera in interphase cells, both chimeras support growth in suspension culture and concanavalin A capping and co- localize with the Con A cap but the Acanthamoeba chimera subsequently disperses more slowly than wild-type myosin and the smooth muscle chimera apparently not at all. Both chimeras also partially rescue chemotaxis. However, neither supports full development. Thus, neither regulation of myosin activity, nor regulation of myosin polymerization nor bipolar filaments is required for many functions of Dictyostelium myosin II and there may be no specific sequence required for localization of myosin to the cleavage furrow. ----------------------------------------------------------------------------- Macromolecular Architecture in Eukaryotic Cells Visualized by Cryo-Electron Tomography Ohad Medalia, Igor Weber, Achilleas S. Frangakis, Guenther Gerisch and Wolfgang Baumeister Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany Science, in press. Abstract Electron tomography of vitrified cells is a non-invasive 3-dimensional imaging technique which opens up new vistas for exploring the supramolecular organization of the cytoplasm. We applied this technique to Dictyostelium cells focusing on the actin cytoskeleton. In actin networks reconstructed without prior removal of membranes or extraction of soluble proteins, the crosslinking of individual microfilaments, their branching angles and membrane attachment sites can be analyzed. At a resolution of 5 to 6 nm, single macromolecules with distinct shapes, such as the 26S proteasome, can be identified in an unperturbed cellular environment. ----------------------------------------------------------------------------- Purification and Renaturation of Dictyostelium Recombinant Alkaline Phosphatase by Continuous Elution Electrophoresis Muatasem Ubeidat and Charles L. Rutherford Biology Department, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406 Protein Expression and Purification, In Press Abstract A 1583 bp fragment of Dictyostelium alp cDNA (94% of the gene) was cloned in pET32a+. The enzyme was expressed in an inactive form in the inclusion body of the expression host BL21-CodonPlus"!(DE3)-RIL. The recombinant ALP constituted more than 50% of the total protein in the inclusion body and 25-30% of the total protein in the expression host after 3 h induction with IPTG at 37 C. A continuous elution polyacrylamide gel electrophoresis procedure was used to purify the recombinant enzyme. This technique yielded a homogenous protein that retained enzymatic activity after dialysis without further treatment. A yield of 5 mg per liter of culture broth was obtained with a specific activity of approximately 0.7 nmol/min/mg of protein (0.7 mU/mg). Immunoinhibition studies using a polyclonal antibody produced against the recombinant protein showed complete inhibition of enzymatic activity when the enzyme was preincubated with the antibody at a 1: 1000 dilution. The enzyme exhibited a pH optimum of approximately 9.0. The substrate specificity indicated that the Dictyostelium enzyme is a typical broad range alkaline phosphatase. ----------------------------------------------------------------------------- [End Dicty News, volume 19, number 8]