dictyNews Electronic Edition Volume 27, number 1 July 14, 2006 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. ============= Abstracts ============= Detection of Cytoplasmic Glycosylation Associated with Hydroxyproline Christopher M. West(1), Hanke van der Wel(1), and Ira J. Blader(2) (1)Department of Biochemistry & Molecular Biology, Oklahoma Center for Medical Glycobiology, and the (2)Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, 73104 USA Methods in Enzymology, in press. A special class of glycosylation occurs on a proline residue of the cytoplasmic/nuclear protein Skp1 in the social amoeba Dictyostelium. In order for this glycosylation to occur, the proline must first be hydroxylated by the action of a soluble prolyl 4-hydroxylase acting on the protein. Cytoplasmic prolyl 4-hydroxylases are dioxygen-dependent enzymes that have low affinity for their O2 substrate and therefore have been implicated in O2-sensing in Dictyostelium as well as in vertebrates and invertebrates. The sugar-hydroxyproline linkage has low abundance, is resistant to alkali cleavage and known glycosidases, and does not bind known lectins. However, initial screens for this modification can be made by assessing changes in electrophoretic mobility of candidate proteins after treatment of cells with prolyl hydroxylase inhibitors, and/or by metabolic labeling with [3H]sugar precursors. In addition, cytoplasmic hydroxylation/glycosylation can be assessed by assaying for cytoplasmic glycosyltransferases. Here we describe these methods and examples of their use in analyzing Skp1 glycosylation in Dictyostelium and the apicomplexan Toxoplasma gondii, the causative agent of toxoplasmosis in humans. Submitted by: Chris West [Cwest2@ouhsc.edu] ----------------------------------------------------------------------------- A 60 kDa protein component of the counting factor complex regulates group size in Dictyostelium Debra A. Brock1, Wouter N. van Egmond2, Yousif Shamoo3, R. Diane Hatton1, and Richard H. Gomer1 1Howard Hughes Medical Institute and 3Department of Biochemistry and Cell Biology, MS-140, Rice University, Houston, TX 77005-1892 2Department of Biochemistry University of Groningen Kerklaan 30 9750 AA Haren The Netherlands Eukaryotic Cell, in press Much remains to be understood about how a group of cells or a tissue senses and regulates its size. Dictyostelium cells sense and regulate the size of groups and fruiting bodies using a secreted 450 kDa complex of proteins called counting factor (CF). Low levels of CF result in large groups, and high levels of CF result in small groups. We previously found three components of CF. We describe here a fourth component, CF60. CF60 has similarity to acid phosphatases, although it has very little, if any, acid phosphatase activity. CF60 is secreted by starving cells and is lost from the 450 kDa CF when a different CF component, CF50, is absent. Although we were unable to obtain cells lacking CF60, decreasing CF60 levels by antisense resulted in large groups, and overexpressing CF60 resulted in small groups. When added to wild-type cells, conditioned starvation medium from CF60 overexpressor cells as well as recombinant CF60 caused the formation of small groups. The ability of recombinant CF60 to decrease group size did not require the presence of the CF components CF45-1 or countin, but did require the presence of CF50. Recombinant CF60 does not have acid phosphatase activity, indicating that the CF60 bioactivity is not due to a phosphatase activity. Together, the data suggest that CF60 is a component of CF, and thus this secreted signal has four different protein components. Submitted by: Richard Gomer [richard@rice.edu] ============================================================================== [End dictyNews, volume 27, number 1]