CSM News Electronic Edition Volume 3, number 7 September 3, 1994 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. =========== Abstracts =========== Temesvari, L.A., Rodriguez-Paris, J., Bush, J., Steck, T.L., and Cardelli, J. Characterization of Lysosomal Membrane Proteins of Dictyostelium discoideum: A Complex Population of Acidic Integral Membrane Glycoproteins, Rab GTP-binding Proteins and Vacuolar ATPase Subunits. J. Biol. Chem., in press. Abstract Highly purified lysosomes, prepared by magnetic fractionation of homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypo-osmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25 kd to 100 kD were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5,-diiodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classifed as integral membrane proteins, and were in general, larger than 45 kD and negatively charged due in part to the presence of mannose-6-sulphate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. Finally, based on N-terminal sequence analysis of a major 41 kD lysosome-associated membrane proteins, we cloned a cDNA that encodes a protein (DVA41) highly homolgous to a yeast and a bovine vacuolar ATPase subunit of approximately 41 kD. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development. --------------------------------------------------------------------- ANALYSIS OF SUCCESSIVE ENDOCYTIC COMPARTMENTS ISOLATED FROM DICTYOSTELIUM DISCOIDEUM BY MAGNETIC FRACTIONATION Kathleen V. Nolta, Juan M. Rodriguez-Paris#, Theodore L. Steck* Department of Biochemistry and Molecular Biology The University of Chicago 920 East 58th Street, Chicago, IL 60637 *Corresponding author. Phone: (312) 702-1329; FAX: (312) 702-0439 E-mail: t-steck@uchicago.edu #Present address: Dept. of Microbiology and Immunology, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130 Biochimica et Biophysica Acta, in press Abstract A colloidal iron probe was fed to the amoeba, Dictyostelium discoideum, and chased for different intervals. Successive segments of the endocytic pathway were then isolated magnetically at high yield and purity. There were ~500 endocytic vacuoles per cell; their diameters increased from ~0.1-0.2 um after 3 min of feeding to ~2 um after 15 min of feeding and 60 min of chase. The wave-like progression of ingested probes along the endocytic pathway suggested that the transfer of cargo involved a maturation mechanism rather than the shuttling of cargo between stable compartments. The lifetime of primary pinosomes was calculated to be ~1 sec. Multivesicular bodies were common in the 3-min fraction and abundant in 15 min lysosomes. Alpha- and beta-adaptins of Mr ~89 and ~83 kDa were richer in the 3-min vesicles than in plasma membranes and later endocytic vacuoles. Acid phosphatase, intrinsic vacuole acidity, the vacuolar proton pump protein and pump activity were present at all endocytic stages but rose between the 3 min and 15 min vacuoles and declined thereafter. Bis(monoacylglycero)phosphate or BMP, a lipid characteristic of lysosomes, followed a similar time course; it contributed up to half of the total lipid in lysosomal vacuoles. We conclude that there is both continuity and differentiation along this endocytic pathway. --------------------------------------------------------------------- Developmental regulation of antisense mediated gene silencing in Dictyostelium. May Sadiq#, Martin Hildebrandt$, Markus Maniak and Wolfgang Nellen* Max-Planck-Institut f. Biochemie, D-82152 Martinsried, Germany # Yarmouk University, Irbid, Jordan $ Inst. Pasteur, Paris, France * corresponding author Antisense Research & Development, in press. Abstract In Dictyostelium, the expression of antisense transcripts has been successfully used to reduce or eliminate gene expression. In most cases this occurs on the level of RNA stability resulting in a loss of both sense and antisense transcript accumulation. We here show that the antisense effect is regulated during the developmental cycle, i.e. in certain developmental stages and under certain developmental conditions, complementary RNAs appear not to interact with each other, resulting in a failure to abolish expression of the gene of interest. We find that this is not only the case with artificially introduced antisense constructs but also with the endogenous, antisense regulated PSV-A gene. Our data demonstrate that antisense mediated gene silencing is conferred by a biochemical machinery which is subject to regulation in vivo. The results provide a basis to better understand this machinery and to dissect the components. They may also explain the failure of some antisense experiments in Dictyostelium and possibly in other organisms. --------------------------------------------------------------------- [End CSM-News, volume 3, number 7]