dictyNews Electronic Edition Volume 38, number 13 May 11, 2012 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 ========= Distinct Functional Roles of the two Terminal Halves of Eukaryotic Phosphofructokinase Oscar H. Mart’nez-Costa, Valentina S‡nchez, Antonio L‡zaro, Eloy D. Hern‡ndez, Keith Tornheim and Juan J. Arag—n Biochemical Journal, published Eukaryotic phosphofructokinase, a key regulatory enzyme in glycolysis, has homologous N- and C-terminal domains thought to result from duplication, fusion, and divergence of an ancestral prokaryotic gene. It has been suggested that both the active site and the fructose 2,6-P2 allosteric site are formed by opposing N- and C-termini of subunits oriented antiparallel in a dimer. On the contrary, we show here that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in phosphofructokinase-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro. The C-terminus is not catalytic but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable, fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions. Submitted by Juan Arag—n [juanjose.aragon@uam.es] -------------------------------------------------------------------------------------- EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins Robert J. Huber a and Danton H. OÕDay 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 Cellular Signalling, in press DdEGFL1, a synthetic Epidermal Growth Factor-Like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210 kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210 kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135 kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32 kDa) as potential components of the DdEGFL1 signalling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signalling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signalling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signalling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge. Submitted by Danton H. OÕDay [danton.oday@utoronto.ca] -------------------------------------------------------------------------------------- Nucleoplasmic/nucleolar translocation and identification of a nuclear localization signal (NLS) in Dictyostelium BAF60a/SMARCD1 homologue Snf12 Andrew Catalano a, and Danton H. OÕDay 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 Dictyostelium is a model eukaryote for the study of several cellular processes however comparatively little is known about its nucleolus. Identification of nucleolar proteins is key to understanding this nuclear subcompartment but only four have been identified in Dictyostelium. As discussed in this article, a potential relationship between nucleolar NumA1 and BAF60a/SMARCD1 suggested BAF60a may also reside in the nucleolus. Here we identify BAF60a homologue Snf12 as the fifth nucleolar protein in Dictyostelium. Immunolocalization experiments demonstrate that Snf12 is nucleoplasmic but translocates to nucleoli upon actinomycin-D-induced transcription inhibition (0.05 mg/mL, 4 hours). Translocation was accompanied by a microtubule-independent protrusion of nucleolar Snf12 regions from the nucleus followed by detection of Snf12 in cytoplasmic circles for at least 48 hours. Residues 372KRKR375 are both necessary and sufficient for nucleoplasmic localization of Snf12 and represent a functional nuclear localization signal (NLS), similar to recently identified NLSs in other Dictyostelium proteins. Since nucleolar and nucleoplasmic proteins redistribute during mitosis, we investigated Snf12 dynamics during this time. Dictyostelium undergoes closed mitosis, meaning its nuclear envelope remains intact. Despite this, during metaphase and anaphase Snf12 redistributed throughout the cytoplasm before reaccumulating in the nucleus during telophase, unlike the previously reported nucleoplasmic redistribution of nucleolar NumA1. The nuclear exit of Snf12 was independent of its putative nuclear export signal and not inhibited by exportin inhibition suggesting the redistribution of nuclear proteins during mitosis in Dictyostelium is mediated by other mechanisms. Snf12 is the second Dictyostelium nucleolar protein for which its dynamics during mitosis have been investigated. Submitted by Danton H. OÕDay [danton.oday@utoronto.ca] ============================================================== [End dictyNews, volume 38, number 13]