Dicty News Electronic Edition Volume 17, number 12 November 17, 2001 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. ============= Abstracts ============= CREATION OF AN ALLOSTERIC PHOSPHOFRUCTOKINASE STARTING WITH A NONALLOSTERIC ENZYME: THE CASE OF DICTYOSTELIUM DISCOIDEUM PHOSPHOFRUCTOKINASE Beln Santamara, Antonio M. Estvez, Oscar H. Martnez-Costa and Juan J. Aragn Departamento de Bioqumica and Instituto de Investigaciones Biomdicas Alberto Sols UAM-CSIC, Facultad de Medicina de la Universidad Autnoma, 28029 Madrid, Spain J. Biol. Chem., accepted for publication, available on line at www.jbc.org Abstract An allosteric phosphofructokinase (PFK) was created by sequence manipulation of the nonallosteric enzyme from the slime mold Dictyostelium discoideum (DdPFK). Most amino acid residues proposed as important for catalytic and allosteric sites are conserved in DdPFK except for a few of them, and their reversion did not modify its kinetic behavior. However, deletions at the unique C-terminal extension of this PFK produced a markedly allosteric enzyme. Thus, a mutant lacking the last 26 C-terminal residues exhibited hysteresis in the time course, intense cooperativity (nH = 3.8) and a 200-fold decrease in the apparent affinity for fructose 6-phosphate (S0.5 = 4500 mM), strong activation by fructose 2,6-bisphosphate (Kact = 0.1 mM) and fructose 1,6-bisphosphate (Kact = 40 mM), dependence on enzyme concentration, proton inhibition and subunit association- dissociation in response to fructose 6-phosphate, versus the nonhysteretic and hyperbolic wild-type enzyme (nH = 1.0; Km = 22 mM) that remained as a stable tetramer. Systematic deletions and point mutations at the C-tail region of DdPFK identified the last C-terminal residue, Leu834, as critical to produce a nonallosteric enzyme. All allosteric mutants were practically insensitive to MgATP inhibition, suggesting that this effect does not involve the same allosteric transition as that responsible for fructose 6-phosphate cooperativity and fructose bisphosphate activation. ----------------------------------------------------------------------------- The Dictyostelium LvsA Protein is Localized on the Contractile Vacuole and is Required for Osmoregulation. Noel J. Gerald, Michael Siano and Arturo De Lozanne Section of Molecular Cell & Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712. Accepted into Traffic LvsA is a Dictyostelium protein that is essential for cytokinesis and that is related to the mammalian beige/LYST family of proteins. To better understand the function of this novel protein family we tagged LvsA with GFP using recombination techniques. GFP-LvsA is primarily associated with the membranes of the contractile vacuole (CV) system and it also has a punctate distribution in the cytoplasm. Two markers of the Dictyostelium CV, the vacuolar proton pump and calmodulin, show extensive colocalization with GFP-LvsA on CV membranes. Interestingly, the association of lvsA with CV membranes occurs only during the discharge phase of the vacuole. In LvsA mutants the CV becomes disorganized and calmodulin dissociates from the CV membranes. Consequently, the CV is unable to function normally, it can swell but seems unable to discharge and the LvsA mutants become osmosensitive. These results demonstrate that LvsA can associate transiently with the CV membrane compartment and that this association is necessary for the function of the CV during osmoregulation. This transient association with specific membrane compartments may be a general property of other BEACH-domain containing proteins. ----------------------------------------------------------------------------- PCR-mediated generation of a gene disruption construct without the use of DNA ligase and plasmid vectors Hidekazu Kuwayama, Shinji Obara, Takahiro Morio, Mariko Katoh, Hideko Urushihara and Yoshimasa Tanaka Institute of Biological Sciences,0University of Tsukuba,0Tsukuba, Ibaraki 305-8572, Japan. Nucleic Acid Research, in press Abstract We introduce a PCR-based procedure for generating a gene disruption construct. This method depends on DNA fragment fusion by PCR technique and requires only two steps of PCR to obtain a sufficient amount of the gene disruption construct for one transformation experiment. The first step involves three separate PCR syntheses of a selectable marker cassette and the 5'- and 3'- regions of a target gene. Of the four primers used in the amplification of the 5 - and 3 -regions of the target gene, two primers placed proximal to the site of the marker cassette are designed to have sequence tags complementary to the 5 - or 3 - side of the marker cassette. The two primers used in the PCR synthesis of the marker cassette are complementary to the tagged primers. By fusion PCR, the 5 - and 3 - PCR products are linked to the marker cassette via the regions of tagged primers which overlap. A sufficient amount of the disruption construct can be directly amplified with the outermost primers. This method is simple, rapid, and relatively inexpensive. In addition, there is the freedom of attaching long flanking regions with any selectable marker cassette. ----------------------------------------------------------------------------- Dictyostelium LvsB Mutants Model the Lysosomal Defects Associated with Chediak-Higashi Syndrome Edward Harris1, Ning Wang2, Wei-l Wu2, Alisha Weatherford1, Arturo De Lozanne2, and James Cardelli1* 1Department of Microbiology and Immunology, LSU Health Sciences Center, Shreveport, LA 71130 2Department of Molecular Cell and Developmental Biology and Institute for Cell and Molecular Biology, University of Texas, Austin, TX 78712 Accepted Molecular Biology of the Cell ABSTRACT Chediak-Higashi Syndrome (CHS) is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans (Lysosomal-Trafficking Regulator) or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of D. discoideum contains 6 genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes, lvsA (large volume sphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE and lvsF. Of all these, only lvsA and lvsB mutants displayed interesting phenotypes in our assays. LvsA-null cells exhibited defects in phagocytosis, and contained abnormal looking contractile vacuole membranes. Loss of LvsB, the Dictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis and fluid phase exocytosis were normal in lvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal alpha-mannosidase were normal, although lvsB mutants inefficiently retained alpha-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes in lvsB-null cells, suggesting LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis. ----------------------------------------------------------------------------- On the Evolutionary Conservation of the Cell Death Pathway: Mitochondrial Release of an Apoptosis-inducing Factor during Dictyostelium discoideum Cell Death Damien Arnoult,* Irene Tatischeff, Jerome Estaquier,* Mathilde Girard, Franck Sureau, Jean Pierre Tissier, Alain Grodet,* Marc Dellinger, FranoisTraincard, Axel Kahn, Jean-Claude Ameisen,* and Patrice Xavier Petit # Mol. Bio. Cell 12, 3016-3030 Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mito-chondrialtransmembrane potential (DYm) that precedes the induction of several apoptosis-likefeatures, including exposure of the phosphatidyl residues at the external surface of the plasmamembrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy,and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyo-stelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms. ----------------------------------------------------------------------------- [End Dicty News, volume 17, number 12]