Dicty News Electronic Edition Volume 12, number 6 February 27, 1999 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============================== Dicty Research Lab Web Pages ============================== If your lab has a web page describing your work using Dictyostelium please be sure that it is listed on the Dicty web site at http://dicty.cmb.nwu.edu/dicty/dicty.html. If your site is not listed send the address of your web site to dicty@nwu.edu. ============== Abstracts ============== Aggregation of Dictyostelium discoideum is dependent on myristoylation and membrane localization of the G protein a-subunit, Ga2. Patsy A. Root, Alison Prince, and Robert E. Gundersen* Biochemistry, Microbiology and Molecular Biology, University of Maine Orono, Maine 04469-5735 J. Cell. Biochem., Accepted for publication Abstract The heterotrimeric G protein, G2, from the eukaryotic organism Dictyostelium discoideum participates in signal transduction pathways which are essential to Dictyostelium s developmental life cycle. G2 is activated by cell surface cAMP receptors and in turn is required for the activation of a host of effectors, including adenylyl cyclase, guanylyl cyclase and phospholipase C. Myristoylation of G protein a-subunits is known to affect a-subunit association with the bg subunits and membrane localization. The putative site for N-terminal myristoylation of Galpha2 was mutated from Gly to Ala (G2A) and expressed in the galpha2-null cell line, MYC2. Transformants expressing Ga2-G2A exhibit physiological and biochemical changes from wild type cells. Ga2-G2A expressing cells fail to rescue the aggregation-minus phenotype of MYC2 cells on developmental agar plates. Ga2-G2A expressing cells are also not chemotactic to cAMP in a standard drop assay. Ga2-WT is found in both the pellet and supernatant fractions following lysis of the cells. Ga2-G2A however is found almost exclusively in the lysate supernatant. Ga2 is radiolabeled upon incubation of cells in [3H]myristate, while Ga2-G2A is not labeled. Examination of activation of the effectors adenylyl cyclase and guanylyl cyclase reveals that Ga2-G2A expressing cells partially activate adenylyl cyclase but show no cAMP-stimulation of guanylyl cyclase. The physiological deviations from wild-type can be explained by the variations in effector activation, possibly due to improper localization of the non-myristoylated Ga2-G2A to the cytosol. ---------------------------------------------------------------------------- Comparison of Base Specificity and Other Enzymatic Properties of Two Protozoan Ribonucleases from Physarum polycephalum and Dictyostelium discoideum Norio INOKUC, Shigeru SAITOH, Hiroko KOBAYASHI, Tadashi ITAGAKI, Takashi KOYAMA, Saburo UCHIYAMA* and Masachika IRIE# Department of Microbiology, College of Pharmacy, Nihon University, Narashinodai 7- 7-1, Funabashi, Chiba 274-0063, Japan *Department of Biology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan #Department of Microbiology, Hoshi College of Pharmacy, Ebara 2-4-41, Shinagawa, Tokyo 142-0063, Japan Biosci. Biotechnol. Biochem., in press Abstract Base specificity and other enzymatic properties of two protozoan RNases, RNase Phyb from a true slime mold (Physarum polycephalum) and RNase DdI from a cellular slime mold (Dictyostelium discoideum), were compared. These two RNases have high amino acid sequence similarity (83 amino acid residues, 46%). The base specificities of two base recognition sites, the B1 site (base recognition site for the base at 5’-side of scissile phosphodiester bond) and the B2 site (base recognition site for the base at 3’-side of the scissile bond) of the both enzymes were estimated by the rates of hydrolysis of 16 dinucleoside phosphates. The base specificities estimated of B1 and B2 sites of RNase Phyb and RNase DdI were A, G, U > C and A, G > C > U, and A, G, U > C and G > U > A, C, respectively. The base specificities estimated from the depolymerization of homopolynucleotides and those from the releases of four mononucleotides upon digestion of RNA coincided well with those of the B2 sites of both enzymes. Thus, in these enzymes, the contribution of the B2 site to base specificity seems to be larger than that of the B1 site. pH-stability, optimum temperature, and temperature stability, of both enzymes are discussed considering that RNase Phyb has one disulfide bridge deleted, compared to the RNase DdI with four disulfide bridges. ---------------------------------------------------------------------------- Regulatory Light Chain Mutations Affect Myosin Motor Function and Kinetics Bernard M. Chaudoir, Patricia A. Kowalczyk and Rex L. Chisholm+ Dept. Of Cell and Molecular Biology, Northwestern University Medical School 303 E. Chicago Ave., Chicago, IL 60611-3008 J. Cell Science, in press. Summary: The actin-based motor protein myosin II plays a critical role in many cellular processes in both muscle and non-muscle cells. Targeted disruption of the Dictyostelium RLC caused defects in cytokinesis and multicellular morphogenesis. In contrast, a myosin heavy chain mutant lacking the RLC binding site and therefore bound RLC showed normal cytokinesis and development. One interpretation of these apparently contradictory results suggests that the phenotypic defects in the RLC null mutant result from mislocalization of myosin caused by aggregation of RLC null myosin. To distinguish this from the alternative explanation that the RLC can directly influence myosin activity, we expressed three RLC point mutations (E12T, G18K and N94A) in a Dictyostelium RLC null mutant. The position of these mutations corresponds to the position of mutations which have been shown to result in familial hypertrophic cardiomyopathy in humans. Analysis of purified Dictyostelium myosin showed that while these mutations did not affect binding of the RLC to the MHC, its phosphorylation by myosin light chain kinase, or regulation of its activity by phosphorylation, they resulted in decreased myosin function. All three mutants showed impaired cytokinesis in suspension, and one produced defective fruiting bodies with short stalks and decreased spore formation. The abnormal myosin localization seen in the RLC null mutant was restored to wildtype localization by expression of all three RLC miutants. Although two of the mutant myosins had wildtype actin-activated ATPase, they produced in vitro motility rates half that of wildtype. N94A myosin showed a five-fold decrease in actin-ATPase and a similar decrease in the rate at which it moved actin in vitro. These results indicate that the RLC can play a direct role in determining the force transmission and kinetic properties of myosin. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 6]