Dicty News Electronic Edition Volume 9, number 8 4 October 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== The patB gene of Dictyostelium discoideum encodes a P-type H+-ATPase isoform essential for growth and development under acidic conditions Barrie Coukell, John Moniakis and Anne Cameron Department of Biology, York University, North York, Ontario, Canada Microbiology, in press Abstract: During growth and early development of Dictyostelium discoideum, the amoebae exhibit transient pH changes in their cytosol (pHi) and external medium which correlate with the extrusion of H+ from the cell by a plasma membrane pump. Moreover, the changes in pHi have been postulated to influence early prestalk/prespore differentiation during development. To learn more about the role of H+ fluxes in Dictyostelium, we cloned and analyzed cDNAs of the gene patB which appears to encode a P-type H+-ATPase. The patB open reading frame specifies a protein (termed PAT2) of 1058 amino acids with a calculated molecular mass of 117, 460 Da. When aligned with other P-type ion transport ATPases, PAT2 showed the greatest amino acid sequence identity with plasma membrane H+-ATPases of plants and fungi and considerably lower identity with other monovalent cation pumps and with Ca2+ pumps. Northern and western analyses revealed that patB is expressed at very low levels in cells growing at neutral pH, but it is up-regulated rapidly and dramatically when the cells are shifted to an acidic medium. Immunofluorescence analysis indicated that PAT2 resides on the plasma membrane. When patB was disrupted by homologous recombination, the cells grew and developed normally at neutral and slightly alkaline pHs but they were unable to grow or develop at pH 5.0, and they slowly died. In growth medium at pH 6.8, patB+ and patB- cells exhibited similar levels of vanadate- sensitive ATPase activity. However, when the cells were shifted to pH 5.0, this activity increased ~2-fold in the control cells but not in the mutant cells. Despite the lower ATPase activity in patB-cells, they showed relatively normal H+ fluxes and only a slight decrease in pHi when incubated in acidic medium. Together, these results suggest that patB specifies an acid-inducible P-type H+-ATPase which is indispensible for the survival of Dictyostelium cells in moderately acidic external environments. ------------------------------------------------------------------------- Null mutations of the Dictyostelium cyclic nucleotide phosphodiesterase gene block chemotactic cell movement in developing aggregates. Richard Sucgang1*, Cornelis J. Weijer2, Florian Siegert3, Jakob Franke1, and Richard H. Kessin1 1Department of Anatomy and Cell Biology, College of Physicians & Surgeons, Columbia University, 630 W 168 Street, New York, NY 10032, USA 2Department of Anatomy and Physiology, Old Medical School, University of Dundee, Dundee DD1 4HN, Scotland 3Zoologisches Institut, Universitat Munchen, Luisenstraáe 14, 80333 Munchen, Germany *Current Address: Department of Biochemistry, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA Abstract Extracellular cAMP is a critical messenger in the multicellular development of the cellular slime mold Dictyostelium discoideum. The levels of cAMP are controlled by a cyclic nucleotide phosphodiesterase (PDE) that is secreted by the cells. The PDE gene (pdsA) is controlled by three promoters that permit expression during vegetative growth, aggregation, and in prestalk cells of the older structures. Targeted disruption of the gene aborts development, and complementation with a modified pdsA restores development. Two distinct promoters must be used for full complementation, and an inhibitory domain of the PDE must be removed. We took advantage of newly isolated PDE-null cells and the natural chimerism of the organism to ask whether the absence of PDE affected individual cell behavior. PDE-null cells aggregated with isogenic wild-type cells in chimeric mixtures, but could not move in a coordinated manner in mounds. The wild-type cells move inward toward the center of the mound, leaving many of the PDE-null cells at the periphery of the aggregate. During the later stages of development, PDE-null cells in the chimera segregate to regions which correspond to the prestalk region and the rear of the slug. Participation in the prespore/spore population returns with the restoration of a modified pdsA to the null cells. ------------------------------------------------------------------------- [End CSM News, volume 9, number 8]