The Gbeta-subunit-encoding gene bpp1 controls cyclic-AMP signaling in Ustilago maydis

In the phytopathogenic fungus Ustilago maydis, fusion of haploid cells is a prerequisite for infection. This process is controlled by a pheromone-receptor system. The receptors belong to the seven-transmembrane class that are coupled to heterotrimeric G proteins. Of four Galpha subunits in U. maydis, only gpa3 has a function during mating and cyclic AMP (cAMP) signaling. Activation of the cAMP cascade induces pheromone gene expression; however, it does not lead to the induction of conjugation tubes seen after pheromone stimulation. To investigate the possibility that a Gbeta subunit participates in pheromone signaling, we isolated the single beta subunit gene, bpp1, from U. maydis. bpp1 deletion mutants grew filamentously and showed attenuated pheromone gene expression, phenotypes associated with deltagpa3 strains. In addition, a constitutively active allele of gpa3 suppressed the phenotype of the bpp1 deletion strains. We suggest that Bpp1 and Gpa3 are components of the same heterotrimeric G protein acting on adenylyl cyclase. Interestingly, while deltagpa3 strains are impaired in pathogenicity, deltabpp1 mutants are able to induce plant tumors. This could indicate that Gpa3 operates independently of Bpp1 during pathogenic development.     

Characterization of a maize G-box binding factor that is induced by hypoxia

G-box cis-acting DNA sequence elements are present in the promoter region of a number of signal-inducible plant genes. In many cases this motif is essential for gene expression. Maize nuclear extracts contain a protein complex that binds specifically to the G-box sequence. Previously, a protein called GF14 was described that is physically associated with the G-box binding complex, but is not a DNA-binding factor in and of itself. This paper reports the isolation of a cDNA encoding a maize G-box binding factor (GBF). The deduced amino acid sequence indicates that maize GBF1 is a basic region-leucine zipper protein. GBF1 binds to the G-box element with specificity similar to that of the binding activity in nuclear extracts. Furthermore, maize GBF1 and the factor detected in nuclear extract are identical in their molecular weight and are immunologically related. GBF1 mRNA accumulates rapidly in hypoxically induced maize cells prior to the increase in Adh1 mRNA levels. Taken together with results that indicate that GBF1 binds to the hypoxia-responsive promoter of maize Adh1, these observations suggest that GBF1 may be one of the factors involved in the activation of Adh1.     

Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF bZIP proteins.

The promoters of a variety of plant genes are characterized by the presence of a G-box (CCACGTGG) or closely related DNA motifs. These genes often exhibit quite diverse expression characteristics and in many cases the G-box sequence has been demonstrated to be essential for expression. The G-box of the Arabidopsis rbcS-1A gene is bound by a protein, GBF, identified in plant nuclear extracts. Here we report the isolation of three Arabidopsis thaliana cDNA clones encoding GBF proteins referred to as GBF1, GBF2 and GBF3. GBF1 and GBF2 mRNA is present in light and dark grown leaves as well as in roots. In contrast, GBF3 mRNA is found mainly in dark grown leaves and in roots. The deduced amino acid sequences of the three cDNAs indicate that each encodes a basic/leucine zipper protein. In addition, all three proteins are characterized by an N-terminal proline-rich domain. Homodimers of the three proteins specifically recognize the G-box motif, with GBF1 and GBF3 binding symmetrically to this palindromic sequence. In contrast, GBF2 binds to the symmetrical G-box sequence in such a way that the juxtaposition of the protein and the DNA element is clearly asymmetric and hence distinct from that observed for the other two proteins. The fact that GBF1, GBF2 and GBF3 possess both distinct DNA binding properties and expression characteristics prompt us to entertain the notion that these proteins may individually mediate distinct subclasses of expression properties assigned to the G-box. Furthermore, we demonstrate that GBF1, GBF2 and GBF3 heterodimerize and these heterodimers also interact with the G-box, suggesting a potential mechanism for generating additional diversity from these GBF proteins.     

GTPgammaS regulation of a 12-transmembrane guanylyl cyclase is retained after mutation to an adenylyl cyclase

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extracellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by guanosine 5'-3-O-(thio) triphosphate (GTPgammaS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCA(kqd) in adenylyl cyclase-defective cells restored the phenotype of the mutant. GTPgammaS stimulated the adenylyl cyclase activity of DdGCA(kqd) with properties similar to those of the wild-type enzyme (decrease of K(m) and increase of V(max)), demonstrating that GTPgammaS stimulation is independent of substrate specificity. Furthermore, GTPgammaS activation of DdGCA(kqd) is retained in several null mutants of Galpha and Gbeta proteins, indicating that GTPgammaS activation is not mediated by a heterotrimeric G-protein but possibly by a monomeric G-protein.     

Control of early gene expression in Dictyostelium

We have examined the expression of a cAMP pulse-repressed and two cAMP pulse-induced genes in response to cAMP and caffeine under a number of different physiological conditions, and in several classes of development mutants altered in cAMP-mediated signal transduction pathways. The data presented help characterize the mutants with regard to early gene expression. Analysis of the data indicates that full induction of the pulse-induced or repression of the pulse-repressed genes requires cycles of activation and adaptation of the cAMP receptor but does not require a rise in intracellular cAMP. Comparison of the results obtained between different mutant classes suggests that repression and activation of the two classes of genes can be uncoupled, implying that different intracellular mechanisms control these processes. In addition, we examined the effects of caffeine and show that it can induce pulse-induced mRNA accumulation in the absence of cAMP.