Dicty News Electronic Edition Volume 21, number 15 November 7, 2003 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 ============= Initiation of Mucin-type O-Glycosylation in Dictyostelium is Homologous to the Corresponding Step in Animals and is Important for Spore Coat Function* Fei Wang½, Talibah Metcalf½¦, Hanke van der Wel½¦, and Christopher M. West½¦ From the ½Department of Anatomy & Cell Biology, College of Medicine, 1600 SW Archer Road, University of Florida, Gainesville, FL 32610-0235, and the ¦Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 USA J. Biol. Chem., in press Like animal cells, many unicellular eukaryotes modify mucin-like domains of secretory proteins with multiple O-linked glycans. Unlike animal mucin-type glycans, those of some microbial eukaryotes are initiated by alpha-linked GlcNAc rather than alpha-GalNAc. Based on sequence similarity to a recently cloned soluble polypeptide hydroxyproline GlcNAc-transferase that modifies Skp1 in the cytoplasm of the social amoeba Dictyostelium, we have identified an enzyme, pp alpha-GlcNAc-T2, that attaches GlcNAc to numerous secretory proteins in this organism. Unlike the Skp1 GlcNAc-transferase, pp alpha-GlcNAc-T2 is predicted to be a type 2 transmembrane protein. A highly purified, soluble, recombinant fragment of pp alpha-GlcNAc-T2 efficiently transfers GlcNAc from UDP-GlcNAc to synthetic peptides corresponding to mucin-like domains in two proteins which traverse the secretory pathway. pp alpha-GlcNAc-T2 is required for addition of GlcNAc to peptides in cell extracts and to the proteins in vivo. Mass spectrometry and Edman degradation analyses show that pp alpha-GlcNAc-T2 attaches GlcNAc in alpha-linkage to the Thr-residues of all the synthetic mucin-repeats. pp alpha-GlcNAc-T2 is encoded by the previously described modB-locus defined by chemical mutagenesis, based on sequence analysis and complementation studies. This finding establishes that the many phenotypes of modB-mutants, including a permeability defect in the spore coat, can now be ascribed to defects in mucin-type O-glycosylation. A comparison of the sequences of pp alpha-GlcNAc-T2 and the animal pp alpha-GalNAc-transferases reveals an ancient common ancestry indicating that, despite the different N-acetylhexosamines involved, the enzymes share a common mechanism of action. Ê Submitted by: Chris West [Christopher-West@ouhsc.edu] ----------------------------------------------------------------------------- Involvement of the AP-1 adaptor complex in early steps of phagocytosis and macropinocytosis Yaya Lefkir, Marilyne Malbouyres, Daniel Gotthardt, Adrian Ozinsky, Sophie Cornillon, Franz Bruckert, Alan A. Aderem, Thierry Soldati, Pierre Cosson, and Franois Letourneur MBC, in press The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1- cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60% indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1- cells is more affected for large rather than small particles and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1- cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation. Submitted by: Francois Letourneur [f.letourneur@ibcp.fr] ----------------------------------------------------------------------------- Proteomics opens doors to the mechanisms of developmentally regulated secretion Stephen Alexander*, Supriya Srinivasan+ and Hannah Alexander* *Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400 +Gladstone Institute of Cardiovascular Disease, University of California-San Francisco, San Francisco, CA 94141 Molecular and Cellular Proteomics, in press. The program of multicellular development in Dictyostelium discoideum culminates with the assembly of a rugged, environmentally resistant spore coat around each spore cell. After synthesis, the proteins that will constitute the coat are stored in prespore vesicles (PSVs) until an unknown developmental signal triggers the PSVs to move to the cell surface where they fuse with the plasma membrane and secrete their cargo by exocytosis. These events occur synchronously in 80% of the cells in each developing multicellular aggregate, and thus the system offers a unique opportunity to study the developmental regulation of protein secretion in situ. Proteomic analysis of purified PSVs identified many of the constituent proteins, which in turn has lead to novel hypotheses and new experimental avenues regarding the molecular mechanisms regulating secretion from the PSVs. Submitted by: Hannah Alexander [AlexanderH@missouri.edu] =============================================================================== [End Dicty News, volume 21, number 15]