ORIGINAL PAPER E. Espagne á P. BalhadeÁre á J. BeÂgueret á B. Turcq Reactivity in vegetative incompatibility of the HE T-E protein of the fungus Podospora anserina is dependent on GTP-binding activity and a WD40 repeated domain Received: 20 January 1997 / Accepted: 8 June 1997 Abstract The het-e gene of the ®lamentous fungus Podospora anserina is involved in vegetative incompati- bility. Co-expression of antagonistic alleles of the un- linked loci het-e and het-c triggers a cell death reaction that prevents the formation of viable heterokaryons between strains that contain incompatible combinations of het-c and het-e alleles. The het-e1 A gene encodes a polypeptide that contains a putative GTP-binding site and WD40 repeats. The role of these two domains in the reactivity of the HET-E protein in incompatibility was analyzed. An in vitro assay con®rmed that the ®rst do- main is functional and can bind GTP and not ATP, suggesting that GTP-binding is essential for triggering the incompatibility reaction. The relationship between the number of WD40 repeats and the reactivity of the protein in incompatibility was investigated by estimating this number in dierent wild-type and mutant het-e alleles. It was deduced that reactive alleles contain a minimal number of ten WD40 repeats. These results demonstrate that the reactivity of the HET-E protein depends on two functional elements, a GTP-binding domain and several WD40 repeats. These motifs are present in separate polypeptides in trimeric G proteins, suggesting that HET-E polypeptides are also involved in signal transduction. Disruption of the het-e locus does not impair the phenotype of strains but DNA hybrid- ization analyses revealed that het-e may belong to a multigenic family. Key words WD40 repeat á GTP-binding á Vegetative incompatibility á Filamentous fungi á Podospora anserina Introduction The WD40 repeat is widespread in eukaryotic proteins (Van Der Voorn and Ploegh 1992). This domain was ®rst identi®ed in the b-subunit of animal heterotrimeric G proteins (Fong et al. 1986). It was then also found in many other proteins involved in various cellular functions, and such as signal transduction, RNA processing, gene regu- lation, regulation of cytoskeleton assembly and vesicular tracking (Neer et al. 1994). Recently, it has been iden- ti®ed in some prokaryotic proteins (Janda et al. 1996). A protein belonging to this WD40 family was found to be encoded by the het-e gene of the ®lamentous fun- gus Podospora anserina (Saupe et al. 1995). This gene is involved in vegetative incompatibility, a lethal reaction which is triggered as the consequence of the co-expres- sion in heterokaryotic cells of antagonistic alleles of the het-e locus and of the unlinked het-c locus. This protein shows some unusual characteristics for a protein that belongs to the WD40 family. The N-terminal region of the protein contains a putative GTP-binding domain. Such a motif has not yet been described for any protein of the WD40 family. The C-terminal region of the pro- tein contains from 3 to 12 WD40 repeats, depending on the het-e allele considered. Such variation in the num- bers of WD40 repeats within a protein has not been previously described. Furthermore, the amino acid se- quence is highly conserved between the dierent repeats, while sequence identity usually does not exceed 20±30% in other proteins of the WD40 family. We have examined the role of these two domains in the reactivity of the HET-E protein in incompatibility reactions. Using an in vitro binding assay, it has been demonstrated that the putative GTP-binding domain is functional and does not bind ATP. A mutation in the P- loop abolishes GTP-binding and results in an het-e allele which is no longer reactive in incompatibility. This suggested that the GTP-binding domain is functional and necessary for triggering cell death. The role of WD40 repeats has been studied by estimating the Mol Gen Genet (1997) 256: 620±627 Ó Springer-Verlag 1997 Communicated by C. A. M. J. J. van den Hondel E. Espagne á P. BalhadeÁre á J. BeÂgueret á B. Turcq (&) Laboratoire de GeÂneÂtique MoleÂculaire des Champignons Filamenteux, UPR CNRS 9026, 1 rue Camille Saint-SaeÈns, F-33077 Bordeaux Cedex, France