Academic journal article Genetics

Three [Alpha]-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria Macrospora

Academic journal article Genetics

Three [Alpha]-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria Macrospora

Article excerpt

ABSTRACT

Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different α-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Δgsa1, Δgsa2, and Δgsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Gα-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Δsa1Δsa2 and Δsa1Δsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Gα-subunits, two recently generated Δpre strains were crossed with all Δsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three ΔsaΔgsac1 double mutants and one Δsa2Δsa3Δgsac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Gα-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

IN eukaryotes, heterotrimeric GTP-binding proteins consisting of α-, β-, and γ-subunits interact with activated heptahelical transmembrane receptors (G protein-coupled receptors, GPCRs) and transduce various environmental signals to stimulate morphogenesis and cellular response. Upon activation by an extracellular signal, the receptor promotes the exchange of GDP for GTP on the Ga-subunit of the heterotrimeric G protein. This in turn leads to the dissociation of Ga from the βγ-complex and each complex can bind and regulate effectors that then can propagate signals into the cell (Hamm 1998; Lengeler et al. 2000). During evolution, G protein subunit genes have expanded enormously in number and diversity. The most complex situation is found in the genome of humans where 27 different genes encoding for Ga-subunits are found (AlbertandRobillard 2002).Onthe basis of sequence similarity, the mammalian Ga-subunits have been divided into four families: (1) Gs activates adenylyl cyclase (AC), (2) Gi inhibits adenylyl cyclase, (3) Gq activates phospholipase C (PLC), and (4) Ga-subunits currently having an unknown function (Hamm 1998). In the genome of the yeast Saccharomyces cerevisiae, only two genes for Ga-subunits (GPA1 and GPA2) have been detected and these are known to play significant roles in mating and filamentous growth (K¨bler et al. 1997; Schrick et al. 1997). During sexual development of S. cerevisiae, two haploid mating types, a and a, communicate via pheromones. While a-cells express genes for a lipopeptide pheromone (a-factor) and the GPCR Ste2p sensing the extracellular a-pheromone, a-cells express genes for a peptide pheromone(a-factor) and theGPCR Ste3p sensing the a-factor. In both cell types, Ste2p and Ste3p are coupled to Gpa1p, one of the two Ga-subunits that forms a conventional heterotrimeric G protein with bg-subunits Ste4p/18p (Dohlman andThorner 2001). Recent studies by Slessareva et al. (2006) revealed a new function for Gpa1p, when they discovered that this Gasubunit not only is located at the plasma membrane, but also is present at the endosomes where it stimulates phosphoinositide 3-kinase (PI3K) to produce PI 3- phosphate. …

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