Molecular genetic analysis of two G alpha protein subunits in Dictyostelium.
In Dictyostelium, chemotaxis to folate during growth and cAMP during aggregation is controlled via cell surface receptors. To study the role of two G alpha proteins (G alpha 1 and G alpha 2) in these responses, we examined the physiological and biochemical effects of null mutations caused by antisense mutagenesis and gene disruptions. Disruption of G alpha 2 results in an aggregation-deficient phenotype and a loss of cAMP receptor-mediated functions, including activation of adenylate cyclase, guanylate cyclase, and gene expression and in a loss of GTP-mediated decrease in receptor affinity for cAMP, but it has no effect on chemotaxis to folate or folate activation of guanylate cyclase. These phenotypes can be rescued by a vector expressing G alpha 2, suggesting G alpha 2 is coupled to a cAMP receptor but not to folate receptors. Loss of G alpha 1 expression resulted in no visible growth or developmental phenotype, including cAMP- and folate-stimulated responses, suggesting G alpha 1 function is either not essential under standard laboratory conditions or is encoded by multiple genes. Availability of null mutations provides suitable genetic backgrounds for expressing mutant G alpha protein subunits which can then be used to examine the physiological roles of G alpha 1 and G alpha 2. Construction of these gene disruptions was facilitated by using the auxotrophic marker THY1, which allowed for selection of single-copy insertions into the genome.
Kumagai
A
A
Department of Biology, University of California, La Jolla 92093.
Hadwiger
J A
JA
Pupillo
M
M
Firtel
R A
RA
D015139:Blotting, Southern; D015153:Blotting, Western; D002633:Chemotaxis; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation; D005492:Folic Acid / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005800:Genes, Fungal; D005822:Genetic Vectors; D006162:Guanylate Cyclase / Q000378:metabolism; D009154:Mutation; D015398:Signal Transduction
D005656:Fungal Proteins; D005492:Folic Acid; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D006162:Guanylate Cyclase; D006152:Cyclic GMP