dictyNews Electronic Edition Volume 40, number 6 February 21, 2014 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 ========= Glycosylation of Skp1 affects its conformation and promotes binding to a model F-box protein M. Osman Sheikh, Christopher M. Schafer, John T. Powell, Karla K. Rodgers, Blaine H. M. Mooers, and Christopher M. West Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 975 NE 10th St., BRC 415, OUHSC, Oklahoma City, OK 73104 USA telephone 1-405-271-4147; email Cwest2@ouhsc.edu Biochemistry, in press In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline-143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2-regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies. Interaction studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1 revealed preferential binding to the glycosylated isoforms of Skp1. This difference correlated with increased alpha-helical and decreased beta-sheet content of glycosylated Skp1s based on circular dichroism, and increased folding order based on small-angle X-ray scattering. A comparison of the molecular envelopes of fully glycosylated Skp1 and the apoprotein indicated that both isoforms exist as an anti-parallel dimer that is more compact and extended in the glycosylated state. Analytical gel filtration and chemical cross-linking studies showed an increasing tendency of less modified isoforms to dimerize. Considering that regions of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds when bound to F-box proteins, we propose that glycosylation, which occurs adjacent to the F-box binding site, influences the spectrum of energetically similar conformations which vary inversely in their propensity to dock with Fbs1 or another Skp1. Glycosylation may thus influence Skp1 function by modulating F-box protein binding in cells.. Submitted by Chris West [Cwest2@ouhsc.edu] --------------------------------------------------------------------------- Novel Regulation of Skp1 by the Dictyostelium AgtA alpha-Galactosyltransferase Involves the Skp1-Binding Activity of its WD40-Repeat Domain Christopher M. Schafer, M. Osman Sheikh, Dongmei Zhang, and Christopher M. West Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, 975 NE 10th St., BRC 415, OUHSC, Oklahoma City, OK 73104 USA telephone 1-405-271-4147; email Cwest2@ouhsc.edu J. Biol. Chem., in press. The role of Skp1 as an adaptor protein that links Cullin-1 to F-box proteins in E3 Skp1/Cullin-1/F-box protein (SCF) ubiquitin ligases is well characterized. In the social amoeba Dictyostelium and probably many other unicellular eukaryotes, Skp1 is modified by a pentasaccharide attached to a hydroxyproline near its C-terminus. This modification is important for oxygen-sensing during Dictyostelium development and is mediated by a HIF-alpha type prolyl 4-hydroxylase and five sequentially acting cytoplasmic glycosyltransferase activities. Gene disruption studies show that AgtA, the enzyme responsible for addition of the final two galactose residues, in alpha-linkages to the Skp1 core trisaccharide, is unexpectedly critical for oxygen-dependent terminal development. AgtA possesses a WD40-repeat domain C-terminal to its single catalytic domain and, by use of domain deletions, binding studies, and enzyme assays, we find that the WD40-repeats confer a salt-sensitive second-site binding interaction with Skp1 that mediates novel catalytic activation in addition to simple substrate recognition. In addition, AgtA binds similarly well to precursor isoforms of Skp1 by a salt-sensitive mechanism that competes with binding to an F-box protein and recognition by early modification enzymes, and the effect of binding is diminished when AgtA modifies Skp1. Genetic studies show that loss of AgtA is more severe when an earlier glycosylation step is blocked, and overexpressed AgtA is deleterious if catalytically inactivated. Together, the findings suggest that AgtA mediates non-enzymatic control of unmodified and substrate precursor forms of Skp1 by a binding mechanism that is normally relieved by switch-like activation of its glycosylation function. Submitted by Chris West [Cwest2@ouhsc.edu] --------------------------------------------------------------------------- Role of PKD2 in Rheotaxis in Dictyostelium Lima WC, Vinet A, Pieters J, Cosson P. PLoS One. 2014 Feb 10;9(2):e88682. The sensing of mechanical forces modulates several cellular responses as adhesion, migration and differentiation. Transient elevations of calcium concentration play a key role in the activation of cells following mechanical stress, but it is still unclear how eukaryotic cells convert a mechanical signal into an ion flux. In this study, we used the model organism Dictyostelium discoideum to assess systematically the role of individual calcium channels in mechanosensing. Our results indicate that PKD2 is the major player in the cell response to rheotaxis (i.e., shear-flow induced mechanical motility), while other putative calcium channels play at most minor roles. Mutant pkd2 KO cells lose the ability to orient relative to a shear flow, whereas their ability to move towards a chemoattractant is unaffected. PKD2 is also important for calcium-induced lysosome exocytosis: WT cells show a transient, 2-fold increase in lysosome secretion upon sudden exposure to high levels of extracellular calcium, but pkd2 KO cells do not. In Dictyostelium, PKD2 is specifically localized at the plasma membrane, where it may generate calcium influxes in response to mechanical stress or extracellular calcium changes. Submitted by Wanessa Lima [wanessa.delima@unige.ch] --------------------------------------------------------------------------- The essential function of Dictyostelium Dgat1 in triglyceride production, but not in ether lipid synthesis, can be substituted by Dgat2 Xiaoli Du1, Cornelia Herrfurth2, Thomas Gottlieb1, Steffen Kawelke2, Kristin Feussner2, Harald RŸhling1, Ivo Feussner2, and Markus Maniak1 Eukaryotic Cell, in press Triacylglycerol (TAG), the common energy storage molecule is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl-coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in Dictyostelium are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the ER-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite to what is seen in mammalian cells. Dictyostelium Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The Dictyostelium Dgat1, however, produces TAG, wax esters, and, most interestingly also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG either through Dgat1 or Dgat2 activity is essential for Dictyostelium to grow on bacteria, their natural food substrate. Submitted by Markus Maniak [maniak@uni-kassel.de] --------------------------------------------------------------------------- SILAC-based proteomic quantification of chemoattractant-induced cytoskeleton dynamics on a second to minute timescale Grzegorz J. Sobczyk, Jun Wang, & Cornelis J. Weijer Nature Communications, in press Cytoskeletal dynamics during cell behaviours ranging from endocytosis and exocytosis to cell division and movement is controlled by a complex network of signalling pathways, the full details of which are as yet unresolved. Here we show that SILAC-based proteomic methods can be used to characterize the rapid chemoattractant-induced dynamic changes in the actinÐmyosin cytoskeleton and regulatory elements on a proteome- wide scale with a second to minute timescale resolution. This approach provides novel insights in the ensemble kinetics of key cytoskeletal constituents and association of known and novel identified binding proteins. We validate the proteomic data by detailed microscopy-based analysis of in vivo translocation dynamics for key signalling factors. This rapid large-scale proteomic approach may be applied to other situations where highly dynamic changes in complex cellular compartments are expected to play a key role. Submitted by Grzegorz Sobczyk [g.j.sobczyk@dundee.ac.uk] ============================================================== [End dictyNews, volume 40, number 6]