CSM News Electronic Edition Volume 3, number 8 September 10, 1994 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. ============== Announcement ============== The next update for the Franke database has been deposited into the archive. This update, update6 is now available for download. Thanks go to Jacob Franke and Richard Sucgang for their efforts to maintain this database! =========== Abstracts =========== Regulatory role of the Ga1 subunit in controlling cellular morphogenesis in Dictyostelium Suranganie Dharmawardhane1, Andrew B. Cubitt, Alexandra M. Clark and Richard A. Firtel2 Department of Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634 telephone: 619-534-2788 fax: 619-534-7073 DEVELOPMENT, IN PRESS SUMMARY To determine the function of the Dictyostelium Ga1 subunit during aggregation and multicellular development, we analyzed the phenotypes of ga1 null cells and strains overexpressing either wild-type Ga1 or two putative constitutively active mutations of Ga1. Strains overexpressing the wild-type or mutant Ga1 proteins showed very abnormal culmination with an aberrant stalk differentiation. The similarity of the phenotypes between Ga1 overexpression and expression of a putative constitutively active Ga1 subunit suggests that these phenotypes are due to increased Ga1 activity rather than resulting from a non-specific interference of other pathways. In contrast, ga1 null strains showed normal morphogenesis except that the stalks were thinner and longer than those of wild-type culminants. Analysis of cell-type-specific gene expression using lacZ reporter constructs indicated that strains overexpressing Ga1 show a loss of ecmB expression in the central core of anterior prestalk AB cells. However, expression of ecmB in anterior-like cells and the expression of prestalk A-specific gene ecmA and the prespore-specific gene SP60/cotC appeared normal. Using a Ga1/lacZ reporter construct, we show that Ga1 expression is cell-type-specific during the multicellular stages, with a pattern of expression similar to ecmB, being preferentially expressed in the anterior prestalk AB cells and anterior-like cells. The developmental and molecular phenotypes of Ga1 overexpression and the cell-type-specific expression of Ga1 suggest that Ga1-mediated signaling pathways play an essential role in regulating multicellular development by controlling prestalk morphogenesis, possibly by acting as a negative regulator of prestalk AB cell differentiation. During the aggregation phase of development, ga1 null cells display a delayed peak in cAMP-stimulated accumulation of cGMP compared to wild-type cells, while Ga1 overexpressors and dominant activating mutants show parallel kinetics of activation but decreased levels of cGMP accumulation compared to that seen in wild-type cells. These data suggest that Ga1 plays a role in the regulation of the activation and/or adaptation of the guanylyl cyclase pathway. In contrast, the activation of adenylyl cyclase, another pathway activated by cAMP stimulation, was unaffected in ga1 null cells and cell lines overexpressing wild-type Ga1 or the Ga1(Q206L) putative dominant activating mutation. However, the Ga1(G45V) putative constitutively active mutation showed significantly reduced adenylyl cyclase activity in response to cAMP. All Ga1 mutant cell lines aggregated normally; however, aggregates of cells expressing Ga1(G45V) developed ring-like structures that then developed a polarity and a small mound-like structure before forming a slug. Immunoprecipitation results suggest that the G45V phenotypes may be due to altered properties of this subunit and its association with the bg subunit. -------------------------------------------------------------------- Assignment of Disulfide Bonds in gp64, a Putative Cell-Cell Adhesion Protein of Polysphondylium pallidum : Presence of Sushi Domains in the Cellular Slime Mold Protein Tamao Saito, Takashi Kumazaki, and Hiroshi Ochiai Department of Biology, Faculty of Science, and the Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060, Japan J. Biol. Chem. in press Summary The 64-kDa membrane-bound glycoprotein of the cellular slime mold Polysphondylium pallidum (referred to as gp64), seems to be implicated in cell-cell adhesion. Previously we have isolated a full-length gp64 cDNA, determined its nucleotide sequence and found that all cysteine residues in the protein are involved in the formation of disulfide bonds. The disulfide arrangement of the 36 cysteines in gp64 was established by analysis of proteolytically cleaved protein and sequence analysis of cystine-containing fragments. Since gp64 has 36-Cys re sidues, 18 disulfide bonds must exist and the positions of 15 of them were determined. The 15 disulfide bonds in gp64 constitute five characteristic, so-called Sushi domains. In a Sushi domain, the first Cys in a sequence is connected to the third one and the second Cys to the fourth one. mAb 293 blocks cell adhesion of Polysphondylium pallidum and its inhibitory activity were cancelled with L-fucose (Toda, K., Tharanathan, R. N., Bozzaro, S., & Gerisch, G. (1984) Eur.J. Biol. 143,477-481 ). Thus, we analyzed the epitope on gp64 and found that mAb 293 bound to the peptide K8r (Ala115-Lys187). This is the first report describing the presence of Sushi domains in a cellular slime mold protein. From these data, gp64 appears to be distinct from all other cell-adhesion proteins described until now. ---------------------------------------------------------------------- ELECTROMAGNETIC PURIFICATION OF ENDOCYTIC VACUOLES AND ACIDOSOMES FROM DICTYOSTELIUM Harish Padh Center for Biotechnology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208, Tel: 708-467-1453, Fax: 708-467-2180, E-mail: padh@nwu.edu ABSTRACT Earlier studies have shown that, in Dictyostelium discoideum, ~90% of the vacuolar proton pump (V-H+-ATPase) activity is present in a buoyant membrane fraction called 'acidosomes'. In the presence of Mg2+, acidosomes and endocytic vacuoles copurified on equilibrium sucrose gradients suggesting their reversible association. The association depended on Mg2+ and cytosolic proteins (1,2). To further characterize the putative association of acidosomes and endocytic vacuoles, cells were fed dextran-coated superparamagnetic iron colloid plus FITC-dextran to load and label their endocytic vacuoles. The endocytic vesicles were then purified ~20-fold at >60% yield by their retention on a column of fine steel wire in an electromagnetic field in the absence of Mg2+. The fraction retained on a magnet column contained only about 5% of total cellular V-H+-ATPase and traces of other organelle markers. In the presence of 1.5 mM Mg2+, however, the retention of V-H+-ATPase as well as FITC-dextran was ~60% with only traces of contaminant markers. When such preparations were washed with buffer lacking Mg2+ while still in the magnetic field, the endocytic marker (FITC-dextran) remained on the column while V-H+-ATPase was eluted selectively. The elute was shown by negative-stain electron microscopy to contain purified acidosomes (saccular membranes studded with V-H+-ATPase). The parent material, recovered from the column in the presence of Mg2+, was rich in endocytic vacuoles bearing colloidal iron. In an electron microscope, the endocytic vacuoles were often seen associated with pump-studded acidosomes. The results independently support and extend earlier observation that acidosomes and endocytic vacuoles physically associate in a Mg2+-dependent manner. In addition, the procedure provides a rapid method to purify acidosomes. -------------------------------------------------------------------- [End CSM News, volume 3, number 8]