The WW domain protein PRO40 is required for fungal fertility and associates with Woronin bodies

Fruiting body formation in ascomycetes is a highly complex process that is under polygenic control and is a fundamental part of the fungal sexual life cycle. However, the molecular determinants regulating this cellular process are largely unknown. Here we show that the sterile pro40 mutant is defective in a 120-kDa WW domain protein that plays a pivotal role in fruiting body maturation of the homothallic ascomycete Sordaria macrospora. Although WW domains occur in many eukaryotic proteins, homologs of PRO40 are present only in filamentous ascomycetes. Complementation analysis with different pro40 mutant strains, using full-sized or truncated versions of the wild-type pro40 gene, revealed that the C terminus of PRO40 is crucial for restoring the fertile phenotype. Using differential centrifugation and protease protection assays, we determined that a PRO40-FLAG fusion protein is located within organelles. Further microscopic investigations of fusion proteins with DsRed or green fluorescent protein polypeptides showed a colocalization of PRO40 with HEX-1, a Woronin body-specific protein. However, the integrity of Woronin bodies is not affected in mutant strains of S. macrospora and Neurospora crassa, as shown by fluorescence microscopy, sedimentation, and immunoblot analyses. We discuss the function of PRO40 in fruiting body formation.     

Swapping functional specificity of a MADS box protein: residues required for Arg80 regulation of arginine metabolism

Arg80 and Mcm1, two members of the MADS box family of DNA-binding proteins, regulate the metabolism of arginine in association with Arg81, the arginine sensor. In spite of the high degree of sequence conservation between the MADS box domains of the Arg80 and Mcm1 proteins (56 of 81 amino acids), these domains are not interchangeable. To determine which amino acids define the specificity of Arg80, we swapped the amino acids in each secondary-structure element of the Arg80 MADS box domain with the corresponding amino acids of Mcm1 and assayed the ability of these chimeras to regulate arginine-metabolic genes in place of the wild-type Arg80. Also performed was the converse experiment in which each variant residue in the Mcm1 MADS box domain was swapped with the corresponding residue of Arg80 in the context of an Arg80-Mcm1 fusion protein. We show that multiple regions of Arg80 are important for its function. Interestingly, the residues which have important roles in determining the specificity of Arg80 are not those which could contact the DNA but are residues that are likely to be involved in protein interactions. Many of these residues are clustered on one side of the protein, which could serve as an interface for interaction with Arg81 or Mcm1. This interface is distinct from the region used by the Mcm1 and human serum response factor MADS box proteins to interact with their cofactors. It is possible that this alternative interface is used by other MADS box proteins to interact with their cofactors.     

Nucleotide sequences recognized by the AGAMOUS MADS domain of Arabidopsis thaliana in vitro

The AGAMOUS gene of Arabidopsis thaliana is a homeotic gene involved in the development of stamens and carpels. This gene encodes a putative DNA-binding protein sharing a homologous region with the DNA-binding domains, MADS boxes, of yeast MCM1 and mammalian SRF. To examine the DNA-binding activity of the AGAMOUS protein, double-stranded oligonucleotides with random sequences of 40 bp in the central region were synthesized and mixed with the AGAMOUS MADS domain overproduced in Escherichia coli . Oligonucleotides which bound to the MADS domain were recovered by repeated immunoprecipitation with an antibody which recognizes the overproduced protein. From a comparison of the recovered DNA sequences, the consensus sequence of the high-affinity binding-sites for the AGAMOUS MADS domain was determined to be 5'-TT(A/T/G) CC(A/T)₆GG(A/T/C)AA-3'. DNase I footprinting and methylation interference experiments showed that the MADS domain binds to this motif. Comparisons with the binding-site sequences of other MADS-box proteins revealed that the MCM1 binding-sites in a-mating type-specific promoters of Saccharomyces cerevisiae show similarities with the binding-site sequence of the AGAMOUS MADS domain. A synthetic MCM1 binding-site in the upstream region of the STE2 gene is recognized by the AGAMOUS MADS domain.