dictyNews Electronic Edition Volume 40, number 27 October 31, 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 Promotes Formation of Skp1/Cullin-1/F-box Protein Complexes in Dictyostelium M. Osman Sheikh*, Yuechi Xu*, Hanke van der Wel*, Paul Walden*, Steven D. Hartsonā, Christopher M. West* *Dept. of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 USA; āDept. of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, OK 74078 USA Molecular & Cellular Proteomics, in press O2-sensing in diverse protozoa depends on the prolyl 4-hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of 5 sugars. In yeast, plants and animals, Skp1 is associated with F-box proteins. The Skp1/F-box protein heterodimer can, for many F-box proteins, dock onto Cullin-1 en route to assembly of the Skp1/Cullin-1/F-box protein/Rbx1 subcomplex of E3SCFUb-ligases. E3SCFUb-ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S-proteasome. In the social amoeba Dictyostelium, we show that O2-availability is rate limiting for hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knock-out mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells that can fully glycosylate Skp1 have greater abundance of an SCF complex containing the Cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, relative to the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA/Skp1CulA complex was also more abundant in glycosylation competent cells. The CulE interactome also includes higher levels of proteasomal regulatory particles when Skp1 is glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding consistent with an impact on the abundance of potential substrate proteins. We propose that O2-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3SCFUb-ligases that regulate O2-dependent developmental progression. Submitted by Chris West [Cwest2@ouhsc.edu] ---------------------------------------------------------------------- Argonaute proteins affect siRNA levels and accumulation of a novel extrachromosomal DNA from the Dictyostelium retrotransposon DIRS-1 Benjamin Boesler1, Doreen Meier1, Konrad U. Fšrstner2, Michael Friedrich1, Christian Hammann3, Cynthia M. Sharma2 and Wolfgang Nellen1,* The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint first authors. 1 Department of Genetics, FB10, Kassel University, Heinrich-Plett-Str. 40, 34132 Kassel, Germany 2 Research Center for Infectious Diseases (ZINF), University of WŸrzburg, Josef-Schneider-Str. 2/Bau D15, 97080 WŸrzburg, Germany 3 Ribogenetics Biochemistry Laboratory, School of Engineering and Science, Molecular Life Sciences Research Center, Jacobs University, Campus Ring 1, DE-28759 Bremen, Germany J. Biol. Chem., accepted doi:10.1074/jbc.M114.612663 The retrotransposon DIRS-1 is the most abundant retroelement in Dictyostelium discoideum and constitutes the pericentromeric heterochromatin of the six chromosomes in Dictyostelium discoideum. The vast majority of cellular siRNAs is derived from DIRS-1, suggesting that the element is controlled by RNAi related mechanisms. We investigated the role of two of the five Argonaute proteins of D. discoideum, AgnA and AgnB, in DIRS-1 silencing. Deletion of agnA resulted in the accumulation of DIRS-1 transcripts, the expression of DIRS-1 encoded proteins and the loss of most DIRS-1 derived secondary siRNAs. Simultaneously, extrachromosomal single stranded DIRS-1 DNA accumulated in the cytoplasm of agnA- strains. These DNA molecules appear to be products of reverse transcription and thus could represent intermediate structures before transposition. We further show that transitivity of endogenous siRNAs is impaired in agnA- strains. The deletion of agnB alone had no strong effect on DIRS-1 transposon regulation. However, in agnA-/agnB- double mutant strains strongly reduced accumulation of extrachromosomal DNA compared to the single agnA- strains was observed. Submitted by Wolfgang Nellen [nellen@uni-kassel.de] ============================================================== [End dictyNews, volume 40, number 27]