CSM News Electronic Edition Volume 8, number 9 April 12, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== The Cytosolic Glycoprotein FP21 of Dictyostelium discoideum is Encoded by Two Genes Resulting in a Polymorphism at a Single Amino Acid Position Christopher M. West, Emil Kozarova and Patana Teng-umnuay Department of Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235 USA Gene, in press. FP21 is a glycoprotein within the cytosolic compartment of Dictyostelium which carries an unusual carbohydrate modification(s) including the sugars fucose, galactose, N-acetyl-glucosamine and xylose. The soluble pool of FP21 from crude extracts resolves chromatographically into two fractions which differ in their glycosylation. Previous gene mapping studies indicating the existence of two loci suggested that the FP21 fractions might be encoded by different genes. To address this issue, the two genes were cloned and sequenced leading to the prediction that the protein products would differ by only a single amino acid, Ser or Ala, at codon 39. Protein sequence data on CNBr fragments of purified FP21 showed that both gene products are found in each fraction of the soluble pool. After further purification, the two fractions were no longer chromatographically resolvable, and there was no evidence for charge heterogeneity as determined by 2-D gel electrophoresis of whole cells. Thus the initial separation of the different soluble subpopulations of this protein appears to be due to distinct molecular complexes, possibly related to differential glycosylation, and is not the result of the genetically-encoded amino acid polymorphism. --------------------------------------------------------------------- Expression of an Arabidopsis Plasma Membrane Aquaporin in Dictyostelium Results in Hypo-osmotic Sensitivity and Developmental Abnormalities Francois Chaumont, William F. Loomis and Maarten J. Chrispeels Department of Biology, University of California, San Diego, La Jolla, CA Proc. Natl. Acad. Sci. in press The rd28 gene of Arabidopsis thaliana encodes a water channel protein or aquaporin of the plasma membrane. We generated a construct in which transcription of the rd28 cDNA is controlled by the Dictyostelium actin 15 regulatory region and transformed it into cells of Dictyostelium discoideum. Transformants grew well in rich medium and contained RD28 protein in their plasma membrane. However, when shifted to low ionic strength buffer, cells expressing rd28 rapidly swelled and burst indicating that the plant aquaporin allowed water to enter rapidly in these amoebae. Since the rate of osmotic lysis was a function of the osmotic pressure of the buffer, this property could be used to assay suspected aquaporins. We also selected transformants carrying a construct in which the regulatory region of the prespore gene cotB was ligated to the rd28 cDNA. These transformants accumulated rd28 mRNA uniquely in prespore cells. When developed on low ionic buffer, they aggregated and formed normally proportioned slugs but failed to form normal fruiting bodies. The number of refractile spores was reduced 20-fold and the stalks were short after development on filters. The consequences of expressing RD28 in prespore cells could be partially overcome by adding 100 mM sorbitol to the buffer to increase the osmolarity. Under these conditions, cotB::rd28 transformants formed more normal fruiting bodies and the number of viable spores was slightly increased. Since prespore cells have to shrink and dehydrate to form spores, it was not unexpected that expression of an aquaporin would disrupt this process, but it was surprising to find that stalk differentiation was also affected by expression of rd28 in prespore cells. It appears that osmotic stress on prespore cells alters their ability to signal terminal differentiation in prestalk cells. --------------------------------------------------------------------- Structural Biology on march 1997, volume 4 number 3, pp. 223-230: The repeating segements of the F-actin cross-linking gelation factor (ABP-120) have an immunoglobulin-like fold. Paola Fucini, Christian Renner, Christoph Herberhold, Angelika A. Noegel and Tad A. Holak. Max Planck Institute for Biochemistry, D-82152 Martinsried, F.R.G. Nature Stuctural Biology 4: 223-230 The 120,000 Mr gelation factor and a-actinin are the most abundant F-actin cross-linking proteins in Dictyostelium discoideum. Both molecules are rod-shaped homodimers. Each monomer chain is comprised of an actin-binding domain and a rod domain. The rod domain of the gelation factor consists of six 100-residue repetitive segments with high internal homology. We have now determined the three-dimensional structure of segment 4 of the rod domain of the gelation factor from D. discoideum using NMR spectroscopy. The segment consists of seven -sheets arranged in an immunoglobulin-like (Ig) fold. This is completely different from the a-actinin rod domain which consists of four spectrin-like -helical segments. The gelation factor is the first example of an Ig-fold found in an actin-binding protein. Two highly homologous actin-binding proteins from human with similar sequences to the gelation factor, filamin and a 280,000 Mr actin-binding protein (ABP-280), share conserved residues that form the core of the gelation factor repetitive segment structure. Thus, the segment 4 structure should be common to this subfamily of the spectrin superfamily. The structure of segment 4 together with previously published electron microscopy data, provide an explanation for the dimerization of the whole gelation factor molecule. ----------------------------------------------------------------------- Evidence for a Recycling Role for Rab7 in Regulating a Late Step in Endocytosis and in Retention of Lysosomal Enzymes in Dictyostelium discoideum Greg Buczynski, John Bush, Linyi Zhang, Juan Rodriguez-Paris, and James Cardelli Department of Microbiology and Immunology and Center for Excellence in Cancer Research, Louisiana State University Medical Center, Shreveport, LA 71130 Mol. Biol. Cell, in press The mammalian small Mr GTPase, Rab7 (Ypt7 in yeast), has been implicated in regulating membrane traffic at post-internalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammlian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, post-lysosomes and maturing phagosomes. Cell-lines were generated that overexpressed Rab7 wild-type (WT), Rab7 Q67L (constitutively active) and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell-line internalized fluid phase markers and latex beads (phagocytosis) at 1/3 the rate of control cells, while Rab7 WT and Rab7 Q67L cell-lines were normal in uptake rates, but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time, and were more slowly exocytosed from Rab7 T22N cells as compared to control cells. Light microscopy indicated that Rab7 expressing cell-lines contained morphologically altered endosomal compartments. Compared to control cells, Rab7 WT and Rab7 Q67L expressing cells contained a reduced number of vesicles the size of post-lysosomes (> > 2.5 microns) and an increased number of smaller vesicles, many of which were non-acidic; in control cells, >90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large vesicles relative to non-acidic vesicles. Radio-label pulse-chase experiments indicated that all the cell-lines processed and targeted lysosomal alpha mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell-lines. Opposite to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted lysosomal hydrolases, while Rab7 WT cells retained these enzymes as compared to control cells. These data support a model that proposes Rab7 may regulate retrograde transport of lysosomal enzymes and the proton pump from post-lysosomes to lysosomes coupled with the efficient release of fluid phase from cells. --------------------------------------------------------------------- [End CSM News, volume 8, number 9]