Dicty News Electronic Edition Volume 13, number 10 October 30, 1999 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/dicty.html" ============= Abstracts ============= LvsA, a protein related to the mouse beige protein, is required for cytokinesis in Dictyostelium. Eunice Kwak*, Noel Gerald*, Denis A. Larochelle*, Kalpa K. Vithalani*, Maria L. Niswonger*, Melinda Maready* and Arturo De Lozanne*@ *Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 and @Section of Molecular Cell & Developmental Biology, University of Texas at Austin, Austin, TX 78712. Molecular Biology of the Cell, in press (December, 1999) ABSTRACT We isolated a Dictyostelium cytokinesis mutant with a defect in a novel locus called large volume sphere A (lvsA). LvsA mutants exhibit an unusual phenotype when attempting to undergo cytokinesis in suspension culture. Early in cytokinesis, they initiate furrow formation with concomitant myosin II localization at the cleavage furrow. However, the furrow is later disrupted by a bulge that forms in the middle of the cell. This bulge is bounded by furrows on both sides, which are often enriched in myosin II. The bulge can increase and decrease in size multiple times as the cell attempts to divide. Interestingly, this phenotype is similar to the cytokinesis failure of Dictyostelium clathrin heavy chain mutants. Furthermore, both cell lines cap ConA receptors, but form only a C-shaped loose cap. Unlike clathrin mutants, lvsA mutants are not defective in endocytosis or development. The LvsA protein shares several domains in common with the molecules beige and Chediak-Higashi syndrome proteins that are important for lysosomal membrane traffic. Thus, based on the sequence analysis of the LvsA protein and the phenotype of the lvsA mutants, we postulate that LvsA plays an important role in a membrane processing pathway that is essential for cytokinesis. ---------------------------------------------------------------------------- Purification and Cloning of TF2: A Novel Protein that binds a Regulatory Site of the gp2 Promoter in Dictyostelium Nikita Warner and Charles L. Rutherford Biology Department, Molecular and Cellular Biology Section, Virginia Polytechnic Institute and State University Blacksburg, VA 24061-0406 Archives of Biochemistry and Biophysics, In Press ABSTRACT The glycogen phosphorylase-2 gene is developmentally regulated and plays a central role in cellular differentiation in Dictyostelium. There are two isozymes of glycogen phosphorylase, GP1 and GP2. Both forms are developmentally regulated; gp1 is expressed in undifferentiated cells and gp2 during differentiation. We report here the identification, purification and cloning of a second gp2 DNA-binding factor called TF2. This protein demonstrates a high specificity for a transcriptional regulatory element, the 5¢ C box. TF2 was first detected with electrophoretic mobility shift assays of DEAE chromatographic fractions of cell-free extracts. The specificity of TF2 for the 5¢ C box was tested by competition analysis using six other oligonucleotides. Purification of TF2 was achieved by ion-exchange chromatography, DNA affinity chromatography, gel filtration chromatography, and preparative SDS-PAGE. SDS-PAGE analysis indicated an apparent subunit molecular weight of 28 kDa. The apparent molecular weight of the native protein as estimated by gel filtration was about 53 kDa. This suggested that TF2 binds gp2 as a homodimer. Southern blot analysis indicated that there is only one form of the tf2 gene. Northern analysis showed little or no expression of tf2 in undifferentiated cells. During development tf2 expression increases up to a maximum at 8 h, then decreases in later stages. Attempts to disrupt the gene suggest that tf2 mutation may be lethal. ---------------------------------------------------------------------------- Differential In Vitro Activation and Deactivation of Cysteine Proteinases Isolated During Spore Germination and Vegetative Growth of Dictyostelium discoideum Dora Cavallo, David Cervi, Todd W. Sands and David A. Cotter Department of Biological Sciences, University of Windsor, Windsor Ontario, Canada, N9B 3P4 Eur. J. Biochem., in press. Summary Acid-activatable cysteine proteinases of D. discoideum were first identified in spore extracts of strain SG1 using gelatin-SDS-PAGE, followed by acid treatments. Here we utilized the technique of acid activation to identify cryptic cysteine proteinases throughout auto-induced and heat- induced spore germination of D. discoideum strain SG2 and SG1. The major acid activatable cysteine proteinase identified in SG2 and SG1 spore extracts was ddCP38 (D. discoideum cysteine proteinase with a molecular weight of 38 kDa) and ddCP48, respectively. Further investigation of these enzymes revealed they were also base de-activatable with a treatment ammonium chloride directly following acid activation. However, the most intriguing observation was the reversibility of the effects of base deactivation on the enzymes following a second treatment with acetic acid. Thus, we hypothesize that, unlike most mammalian cysteine proteinases that generally require the cleavage of a pro-peptide region for activation, these cysteine proteinases of D. discoideum likely undergo reversible conformational changes between latent and active forms. Moreover, we were able to detect these cryptic cysteine proteinases in the vegetative cells and early aggregates of both strains SG1 and SG2. Studies using 4-[(2S,3S)-3-carboxyoxiran-2- ylcarbonyl-L-leucylamido]butylguanidine, a cysteine proteinase inhibitor, revealed that acid activation of a portion of these proteinases was still achievable even after incubation with the inhibitor, further supporting the concept of two stable and reversible conformational arrangements of the enzymes. Thus, we speculate that the pH shuffles that modulate proteinase conformation and activity in vitro may be a reflection of the in vivo regulation of these enzymes via H+-ATPases and ammonia. ---------------------------------------------------------------------------- Circulation of the plasma membrane in Dictyostelium by Carmen Aguado-Velasco and Mark S. Bretscher* MRC Laboratory for Molecular Biology, Hills Road, Cambridge CB2 2QH Molecular Biology of the Cell, in press. ABSTRACT We have developed a fluorimetric assay using the dye FM1-43 to determine the rate at which Dictyostelium amoebae endocytose their surface membrane. Our results show that they do so about once each 4-10 minutes. A clathrin null mutant takes its surface up only about 30% more slowly, showing that this membrane uptake cannot be caused by clathrin-coated vesicles. Surprisingly, Ax2 and its parent NC4, which differ in their rates of fluid phase internalisation by about 60-fold, take up their surfaces at the same rates. These results show that, in axenic cells, the uptake of fluid and of surface area are separate processes. The large activity of this new endocytic cycle in both Ax2 and NC4 amoebae appears capable of delivering sufficient new surface area to advance the cells' fronts during migration. ---------------------------------------------------------------------------- The prespore vesicles of Dictyostelium discoideum: Purification, Characterization and Developmental Regulation Supriya Srinivasan, Hannah Alexander and Stephen Alexander* Division of Biological Sciences University of Missouri Columbia, MO 65211 J. Biol. Chem., in press. Abstract: The coordinate fusion of the prespore vesicles (PSVs) with the plasma membrane at the terminal stage of spore differentiation in Dictyostelium discoideum is an important example of developmentally regulated protein secretion. However, little is known about the composition of the vesicles, the molecular signals regulating secretion or the mechanics of the membrane fusion. Taking a biochemical approach, we purified PSVs from different developmental stages. These preparations are highly enriched for their specific cargo of spore coat proteins while devoid of markers for other cellular compartments. Electron microscopic observations show that the PSV preparations are homogenous, with the soluble spore coat protein PsB/SP85 distributed throughout the lumen and the acid mucopolysaccharide localized in the central core. During development the PSVs increase in size and density concomitant with an increase in their protein cargo. The PSVs contain approximately 80 proteins, and we have identified a PSV-specific GTP-binding protein which may be involved in regulating vesicle fusion. The PSVs are not clathrin coated, and do not contain the SpiA spore coat protein. The PSV preparations are ideal for a global proteome analysis to identify proteins involved in signal reception, vesicle movement, docking and fusion in this developmentally regulated organelle. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 10]