CSM News Electronic Edition Volume 1, number 11 July 31, 1993 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. ======================================================================= Recently Accepted Papers ======================================================================= The actin-binding protein comitin (p24) is a component of the Golgi apparatus Olaf H. Weiner, John Murphy, Michael Schleicher* and Angelika A. Noegel. Max-Planck-Institut fur Biochemie, Am Klopferspitz 18a, 8033 Martinsreid, F.R.G. J. Cell Biology, in press. Summary Comitin (p24) was first identified in Dictyostelium discoideum as a membrane associated protein which binds in gel overlay assays to G- and F-actin. To analyze its actin-binding properties we used purified, bacterially expressed comitin and found that it binds to F-actin in spin down experiments and increases the viscosity of F- actin solutions even under high salt conditions. Immunofluorescence studies, cell fractionation experiments and EM studies of vesicles precipitated with comitin-specific monoclonal antibodies showed that comitin was present in D. discoideum on i) a perinuclear structure with tubular or fibrillary extensions and ii) on vesicles distributed throughout the cell. In immunofluorescence experiments using comitin antibodies NIH 3T3- fibroblasts showed a similar staining pattern as D. discoideum cells. Using bona fide Golgi markers the perinuclear structure was identified as the Golgi apparatus. Based on these results we propose that also in Dictyostelium the stained perinuclear structure is the Golgi apparatus. In vivo the perinuclear structure was found to be attached to the actin and the microtubule network. Alteration of the actin network or depolymerization of the microtubules led to its dispersal into vesicles distributed throughout the cell. These results suggest that the Golgi apparatus in D. discoideum is connected to the actin network by comitin. This protein seems also to be present in mammalian cells. ------------------------------------------------------------------ The heat shock cognate protein from Dictyostelium affects actin polymerization through interaction with the actin-binding protein cap32/34 Ulrike Haus, Paul Trommler, Paul R. Fisher (2), Herbert Hartmann, Friedrich Lottspeich, Angelika A. Noegel and Michael Schleicher (1,3) Max-Planck-Institut fuer Biochemie, Am Klopferspitz 18a, 82152 Martinsried, and (1) Ludwig-Maximilians-Universitaet Muenchen, Institut fuer Zellbiologie, Schillerstra!e 42, 80336 Muenchen, FRG; (2) La Trobe University, Department of Microbiology, Bundoora, Victoria, Australia 3083 EMBO J., In press. Summary During isolation of the F-actin capping protein cap32/34 from Dictyostelium discoideum a 70kDa protein was copurified which by cloning and sequencing was identified as a heat shock cognate protein (hsc70). This protein exhibited a specific and MgATP-dependent interaction with the heterodimeric capping protein. Immunofluorescence studies indicated that hsc70 and the capping protein are localized in the same regions of D. discoideum cells. To investigate the protein/protein interaction in vitro, we expressed all three polypeptides separately in Escherichia coli and performed reconstitution experiments of complete or truncated hsc70 with the 32 kDa and 34kDa subunits of the capping protein. Viscosity measurements and studies on the polymerization kinetics of pyrene-labeled actin showed that hsc70 increased the capping activity of cap32/34 up to ten-fold, whereas hsc70 alone had no effect on actin polymerization. In addition, hsc70 acted as a molecular chaperone by stimulating the refolding of the denatured 32 kDa and 34 kDa subunits of the capping protein. To study the interaction of the two domains of hsc70 with cap32/34, the N- terminal 42 kDa ATPase-region and the C-terminal 30 kDa-tail of hsc70 were expressed separately in E. coli. The 32 kDa and 34 kDa subunits were capable of associating with both domains of hsc70. The ATPase-domain of hsc70, which is structurally related to actin, proved to be responsible for the increased capping activity of cap32/34, whereas the C-terminal tail of hsc70 was involved in refolding of cap32/34. Our data indicate a novel linkage between 70 kDa heat shock proteins and the actin cytoskeleton. A function of the actin-like ATPase domain appears to be the inhibition of actin polymerization in an indirect fashion via an increase in the activity of an F-actin capping protein. --------------------------------------------------------------- The 100kDa F-actin capping protein of Dictyostelium amoebae is a villin prototype (protovillin) A. Hofmann, A.A. Noegel*, L. Bomblies, F. Lottspeich* and M. Schleicher Institute for Cell Biology, Ludwig-Maximilians-University Munich, Schillerstrade 42, 80336 Muenchen, FRG; and *Max-Planck- Institute for Biochemistry, 82152 Martinsried, FRG FEBS-Letters, in press Summary The 100kDa actin-binding protein from Dictyostelium amoebae is an F-actin capping protein that displays neither severing nor crosslinking or nucleating activities (Hofmann et al., (1992) Cell Motil. Cytoskel. 23,133-144). Cloning and sequencing of the gene revealed that the protein is highly homologous to vertebrate villin, a unique component of brush border microvilli and contains six domains fused to a villin-like headpiece domain via a threonine/proline rich neck region. The functional differences and similarities between the 100kDa protein and villin are reflected in the amino acid sequences. We draw from the data the following conclusions: i) The presence of a six domain protein in Dictyostelium suggests that in contrast to the current view gene duplications must have happened before Dictyostelium branched off during evolution. ii) The villin-like molecule in Dictyostelium appears to be a premature villin (protovillin) which is able to cap actin filaments but still lacks the other villin-type actin-binding activities. This renders capping of actin filaments as the evolutionarily oldest function of an F-actin binding protein. ---------------------------------------------------------------------- THE ROLE OF CYCLIC GMP IN REGULATING MYOSIN DURING CHEMOTAXIS OF DICTYOSTELIUM : EVIDENCE FROM A MUTANT LACKING THE NORMAL CYCLIC GMP RESPONSE TO CYCLIC AMP Gang Liu (1), Hidekazu Kuwayama (2), Shuji Ishida (2) and Peter C. Newell (1) (1): Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K. (2) Department of Botany, Faculty of Science, Kyoto University, Kyoto, 606, Japan Summary Evidence has previously been reported that, during chemotaxis of the cellular slime mould Dictyostelium discoideum, cyclic GMP regulates the association of myosin II with the cytoskeleton and that this regulation is effected by inhibiting myosin II heavy chain phosphorylation (Liu & Newell, J.Cell Sci., 90, 123-129, 1988; 98, 483-490, 1991). Here we provide further evidence in support of this hypothesis using a mutant (KI-10) which is defective in chemotaxis and lacks the normal cyclic AMP-induced cyclic GMP response. We found that the cyclic AMP-induced cytoskeletal actin response was similar to the parental strain in this mutant (although showing a slight displacement in the dose-response curve) but the cytoskeletal Myosin II heavy chain response was abolished. Moreover the mutant showed no phosphorylation of myosin II heavy chain in response to cyclic AMP. Compared to the parental strain XP55, the mutant cells contained approximately 40% more protein and their doubling time was 30% longer. These differences could be due to differences in the efficiency of cell division, a process in which the proper regulation of myosin function is essential and in which cyclic GMP may therefore play a role. ------------------------------------------------------------------------ Protein phosphorylation during the process of prestalk-to-stalk conversion in Dictyostelium discoideum Mineko Maeda (1) and Yuzuru Kubohara (2) (1) Department of Biology, College of General Education, Osaka University, Toyonaka, Osaka 560, Japan; Present address: Department of Biology, Center for Molecular Genetics, M-034, La Jolla CA92093, USA (2) Department of Botany, Faculty of Science, Kyoto University, Kyoto 606, Japan Development, Growth and Differ., in press Abstract We have previously shown that 8-bromo cAMP (Br-cAMP) efficiently induces prestalk-to-stalk conversion in in vitro cultures of the cellular slime mold Dictyostelium discoideum, and that protein kinase A (PKA) plays an important role(s) in the conversion process (Kubohara et al. Exp. Cell Res. vol. 207, pp.107-114, 1993). In the present study, we analyzed protein phosphorylation during the Br-cAMP-induced process, and identified two phosphoproteins, p60 and p40, that were specifically induced (phosphorylated) by Br-cAMP. The protein kinase inhibitors, staurosporine and K252a, which blocked the Br cAMP-induced stalk cell formation, also inhibited the phosphorylations of these proteins. These results suggest that p60 and p40 may be the target proteins of PKA and thus be involved in the prestalk-to-stalk conversion. ---------------------------------------------------------------------- [END CSM-News Volume 1, number 11]