dictyNews Electronic Edition Volume 37, number 10 October 21, 2011 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. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Dictyostelium puromycin-sensitive aminopeptidase A is a nucleoplasmic nucleomorphin-binding protein that relocates to the cytoplasm during mitosis Andrew Catalano a, Yekaterina Poloz a, and Danton H. O'Day a,b a Department of Cell and Systems Biology, University of Toronto, 25 Harbord st., Toronto, Ontario, Canada, M5S 3G5 b Department of Biology, University of Toronto at Mississauga, 3359 Mississauga rd. N., Mississauga, Ontario, Canada, L5L 1C6 Histochemistry and Cell Biology, in press Nucleomorphin (NumA1) is a nucleolar/nucleoplasmic protein linked to cell cycle in Dictyostelium. It interacts with puromycin-sensitive aminopeptidase A (PsaA) which in other organisms is a Zn2+-metallopeptidase thought to be involved in cell cycle progression and is involved in several human diseases. Here, we have shown that Dictyostelium PsaA contains domains characteristic of the M1 family of Zn2+ metallopeptidases: a GAMEN motif and a Zn2+ binding domain. PsaA colocalized with NumA1 in the nucleoplasm in vegetative cells and was also present to a lesser extent in the cytoplasm. The same localization pattern was observed in cells from slugs however in fruiting bodies PsaA was only detected in spore nuclei. During mitosis PsaA redistributed mainly throughout the cytoplasm. It possesses a functional nuclear localization signal (680RKRF683) necessary for nuclear entry. To our knowledge this is the first nuclear localization signal identified in a Psa from any organism. Treatment with Ca2+ chelators or calmodulin antagonists indicated that neither Ca2+ nor calmodulin are involved in PsaA localization. These results are interpreted in terms of the inter-relationship between NumA1 and PsaA in cell function in Dictyostelium. Submitted by Danton H. O'Day [danton.oday@utoronto.ca] -------------------------------------------------------------------------------------- The cyclin-dependent kinase inhibitor roscovitine inhibits kinase activity, cell proliferation, multicellular development, and Cdk5 nuclear translocation in Dictyostelium discoideum Robert J. Huber 1,2 and Danton H. O'Day *1,2 1Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada M5S 3G5 2Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6 Journal of Cellular Biochemistry, in press Roscovitine, a cyclin-dependent kinase (Cdk) inhibitor, inhibited kinase activity and the axenic growth of Dictyostelium discoideum at micromolar concentrations. Growth was almost fully rescued in 50 µM and ~50% rescued in 100 µM roscovitine-treated cultures by the over-expression of Cdk5-GFP. This supports the importance of Cdk5 function during cell proliferation in Dictyostelium and indicates that Cdk5 is a primary target of the drug. Roscovitine did not affect the expression of Cdk5 protein during axenic growth but did inhibit its nuclear translocation. This novel result suggests that the effects of roscovitine could be due in part to altering Cdk5 translocation in other systems as well. Kinase activity was inhibited by roscovitine in assays using AX3 whole cell lysates, but not in assays using lysates from Cdk5-GFP over-expressing cells. At higher concentrations, roscovitine impaired slug and fruiting body formation. Fruiting bodies that did form were small and produced relatively fewer spores many of which were round. However roscovitine did not affect stalk cell differentiation. Together with previous findings, these data reveal that roscovitine inhibits Cdk5 during growth and as yet undefined Cdks during mid-late development. Submitted by Danton H. O'Day [danton.oday@utoronto.ca] --------------------------------------------------------------------------------- RpkA, a highly conserved GPCR with a lipid kinase domain, has a role in phagocytosis and anti-bacterial defense Tanja Y. Riyahi, Frederike Frese, Michael Steinert, Napoleon N. Omosigho, Gernot Gloeckner, Ludwig Eichinger, Benoit Orabi, Robin S. B. Williams, Angelika A. Noegel PLoS ONE RpkA (Receptor phosphatidylinositol kinase A) is an unusual seven-helix transmembrane protein of Dictyostelium discoideum with a G protein coupled receptor (GPCR) signature and a C-terminal lipid kinase domain (GPCR-PIPK) predicted as a phosphatidylinositol-4-phosphate 5-kinase. RpkA-homologs are present in all so far sequenced Dictyostelidae as well as in several other lower eukaryotes like the oomycete Phytophthora, and in the Legionella host Acanthamoeba castellani. Here we show by immunofluorescence that RpkA l ocalizes to endosomal membranes and is specifically recruited to phagosomes. RpkA interacts with the phagosomal protein complex V-ATPase as proteins of this complex co-precipitate with RpkA-GFP as well as with the GST-tagged PIPK domain of RpkA. Loss of RpkA leads to a defect in phagocytosis as measured by yeast particle uptake. The uptake of the pathogenic bacterium Legionella pneumophila was however unaltered whereas its intra-cellular replication was significantly enhanced in rpkA-. The difference between wild type and rpkA- was even more prominent when L. hackeliae was used. When we investigated the reason for the enhanced susceptibility for L. pneumophila of rpkA- we could not detect a difference in endosomal pH but rpkA- showed depletion of phosphoinositides (PIP and PIP2) when we compared metabolically labeled phosphoinositides from wild type and rpkA-. Furthermore rpkA- exhibited reduced nitrogen starvation tolerance, an indicator for a reduced autophagy rate. Our results indicate that RpkA is a component of the defense system of D. discoideum as well as other lower eukaryotes. Submitted by Angelika Noegel [noegel@uni-koeln.de] ----------------------------------------------------------------------------------- Identification of a Eukaryotic Reductive Dechlorinase and Characterization of Its Mechanism of Action on Its Natural Substrate Francisco Velazquez,1,* Sew Yu Peak-Chew,1 Israel S. Fernandez,1 Christopher S. Neumann,2 and Robert R. Kay1 *Correspondence: fv@mrc-lmb.cam.ac.uk 1 Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, UK 2 Department of Microbiology, University of Washington, Seattle, WA 98195, USA Chemistry and Biology, in press Chlorinated compounds are important environmental pollutants whose biodegradation may be limited by inefficient dechlorinating enzymes. Dictyostelium amoebae produce a chlorinated alkyl phenone called DIF which induces stalk cell differentiation during their multicellular development. Here we describe the identification of DIF dechlorinase. DIF dechlorinase is active when expressed in bacteria, and activity is lost from Dictyostelium cells when its gene, drcA, is knocked out. It has a Km for DIF of 88 nM and Kcat of 6.7 s1. DrcA is related to glutathione S-transferases, but with a key asparagine-to-cysteine substitution in the catalytic pocket. When this change is reversed, the enzyme reverts to a glutathione S-transferase, thus suggesting a catalytic mechanism. DrcA offers new possibilities for the rational design of bioremediation strategies. Submitted by Francisco Velazquez [fveldua@upo.es] ============================================================== [End dictyNews, volume 37, number 10]