Dicty News Electronic Edition Volume 24, number 16 June 17, 2005 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 Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= The Phosducin-like Protein Phlp1 is Essential for Gbeta-gamma Dimer Formation in Dictyostelium Jaco C. Knol1,3, Ruchira Engel2,3, Mieke Blaauw1, Antonie J.W.G. Visser2 and Peter J.M. van Haastert1 1Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands and 2MicroSpectroscopy Centre, Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands 3 These authors contributed equally to this work Molecular and Cellular Biology, in press Phosducin proteins are known to inhibit G protein-mediated signalling by sequestering Gbeta-gamma subunits. However, Dictyostelium cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signalling. Here we show that GFP-tagged Gbeta and Ggamma subunits exhibit drastically reduced steady state levels and are absent from the plasma membrane in phlp1_ cells. Triton X-114 partitioning suggests that lipid attachment to GFP-Ggamma occurs in wild-type, but not in phlp1_ and gbeta _ cells. Moreover, Gbeta-gamma dimers could not be detected in vitro in co-immunoprecipitation assays with phlp1- cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-Ggamma proteins are present in a complex in wild type cells they are free in phlp1- and gbeta- cells. Collectively, our data strongly suggest the absence of Gbeta-gamma dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a co-chaperone assisting the assembly of Gbeta and Ggamma into a functional Gbeta-gamma complex. Thus, phosducin family proteins may fulfil hitherto unsuspected biosynthetic functions. Submitted by: Peter Van Haastert [p.j.m.van.haastert@rug.nl] ----------------------------------------------------------------------------- Functional analysis of a novel gene, DD3-3, from Dictyostelium discoideum N. Sakuragi, N. Ogasawara, E. Tanesaka, and M. Yoshida, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan Department of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan Biochem. Biophys. Res. Commun. in press A novel gene, DD3-3, from Dictyostelium discoideum has been isolated by an mRNA differential display between a wild-type strain AX2 and a mutant HG794 which is defective in O-glycosylation. Functional analysis of the novel gene, DD3-3, was conducted by preparing a knockout mutant, DD3-3KO, and a GST:DD3-3 fusion protein. The mutant DD3-3KO cells were allowed to develop about 1.5 hours earlier than the wild-type strain AX2 cells. Northern analysis of the knockout mutant cells showed a remarkable downregulation of Reg A, cAMP-dependent phosphodiesterase, and overexpression of protein tyrosine kinase (PTK) during early development and its shutdown during late development. The relationship between O-glycosylation and phosphorylation involving Reg A gene is discussed. Submitted by: Motonobu.Yoshida [yoshida_m@nara.kindai.ac.jp] ============================================================================== [End Dicty News, volume 24, number 16]