dictyNews Electronic Edition Volume 30, number 18 June 6, 2008 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= Antisense RNA Inhibition of the beta Subunit of the Dictyostelium discoideum Mitochondrial Processing Peptidase Induces the Expression of Mitochondrial Proteins Koki Nagayama(1,2), Shiori Itono(2), Takashi Yoshida(2), Sei-ichi Ishiguro(2), Hiroshi Ochiai(2,3), and Tetsuo Ohmachi*(2) (1) Science of Bioresources, United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8551, Japan (2) Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan (3) Creative Research Institutive Sousei (CRIS), Hokkaido University, Sapporo 001-0021, Japan * Correspondingu author: Tel: +81-172-39-3774; Fax: +81-172-39-3750; E-mail: tohmachi@cc.hirosaki-u.ac.jp Bioscience, Biotechnology, and Biochemistry, in press We cloned and characterized a cDNA encoding the Dictyostelium discoideum beta subunit of mitochondrial processing peptidase (Ddb-MPP). Western blot analysis of the mitochondrial subfractions revealed that Ddb-MPP is located in the mitochondrial matrix and membrane, whereas Dda-MPP, another subunit of DdMPP, is located only in the matrix. Although expression of Ddb-MPP mRNA is down-regulated during early development, the level of the Ddb-MPP protein is constant throughout the Dictyostelium life cycle. In a transformant expressing the antisense RNA of the b-MPP gene, unexpectedly, the b-MPP protein increased about 1.8-fold relative to the wild type, and its mRNA increased 4.5-fold. Expression of other mitochondrial proteins, a-MPP and Cox IV, was also induced. These results suggest that antisense RNA inhibition of the beta-MPP gene induces gene expression of mitochondrial proteins, presumably in a retrograde signaling manner. This is the pathway of the transfer of information from mitochondria to the nucleus. Submitted by: Koki Nagayama [i205015@stu.hirosaki-u.ac.jp] -------------------------------------------------------------------------------- Purinergic-mediated Ca(2+) influx in Dictyostelium discoideum. Melanie J. Ludlow#, David Traynor+, Paul R. Fisher§ and Steven J. Ennion# #Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Leicester LE1 9HN, UK. +MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK §Department of Microbiology, La Trobe University, Melbourne VIC 3086, Australia Cell Calcium, in press The presence of five P2X-like genes (p2xA-E) in Dictyostelium suggests that nucleotides other than cAMP may act as extracellular signalling molecules in this model eukaryote. However, p2xA was found to have an exclusively intracellular localisation making it unclear whether Dictyostelium utilise P2 receptors in a manner analogous to vertebrates. Using an apoaequorin expressing strain we show here that Dictyostelium do possess cell surface P2 receptors that facilitate Ca(2+) influx in response to extracellular ATP and ADP (EC(50)=7.5muM and 6.1muM, respectively). Indicative of P2X receptor activation, responses were rapid reaching peak within 2.91+/-0.04s, required extracellular Ca(2+), were inhibited by Gd(3+), modified by extracellular pH and were not affected by deletion of either the single Gbeta or iplA genes. Responses also remained unaffected by disruption of p2xA or p2xE showing that these genes are not involved. Cu(2+) and Zn(2+) inhibited purine-evoked Ca(2+) influx with IC(50) values of 0.9 and 6.3muM, respectively. 300muM Zn(2+) completely abolished the initial large rapid rise in intracellular Ca(2+) revealing the presence of an additional smaller, slower P2Y-like response. The existence of P2 receptors in Dictyostelium makes this organism a valuable model to explore fundamental aspects of purinergic signalling. Submitted by: Steve Ennion [se15@leicester.ac.uk] -------------------------------------------------------------------------------- Vacuole membrane protein 1 is an endoplasmic reticulum protein required for organelle biogenesis, protein secretion and development Javier Calvo-Garrido, Sergio Carilla-Latorre, Francisco Lázaro-Diéguez, Gustavo Egea and Ricardo Escalante Molecular Biology of the Cell, in press Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1- Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1- cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised. Submitted by: Ricardo Escalante [rescalante@iib.uam.es] ============================================================== [End dictyNews, volume 30, number 18]