Cell differentiation during sexual development of the fungus Sordaria macrospora requires ATP citrate lyase activity

During sexual development, mycelial cells from most filamentous fungi diffe rentiate into typical fruiting bodies. Here, we describe the isolation and characterization of the Sordaria macrospora developmental mutant per5, whic h exhibits a sterile phenotype with defects in fruiting body maturation. Cy tological investigations revealed that the mutant strain forms only ascus p recursors without any mature spores. Using an indexed cosmid library, we we re able to complement the mutant to fertility by DNA-mediated transformatio n. A single cosmid clone, carrying a 3.5-kb region able to complement the m utant phenotype, has been identified. Sequencing of the 3.5-kb region revea led an open reading frame of 2.1 kb interrupted by a 66-bp intron, The pred icted polypeptide (674 amino acids) shows significant homology to eukaryoti c ATP citrate lyases (ACLs), with 62 to 65% amino acid identity, and the ge ne was named acl1, The molecular mass of the S. macrospora ACL1 polypeptide is 73 kDa, as was verified by Western blot analysis with a hemagglutinin ( HA) epitope-tagged ACL1 polypeptide, Immunological in situ detection of the HA-tagged polypeptide demonstrated that ACL is located within the cytosol, Sequencing of the mutant acl1 gene revealed a l-nucleotide transition with in the coding region, resulting in an amino acid substitution within the pr edicted polypeptide. Further evidence that ACL1 is essential for fruiting b ody maturation comes from experiments in which truncated and mutated versio ns of the acl1 gene were used for transformation. None of these copies was able to reconstitute the fertile phenotype in transformed per5 recipient st rains, ACLs are usually involved in the formation of cytosolic acetyl coenz yme A (acetyl-CoA), which is used for the biosynthesis of fatty acids and s terols, Protein extracts from the mutant strain showed a drastic reduction in enzymatic activity compared to values obtained from the wild-type strain . Investigation of the time course of ACL expression suggests that ACL is s pecifically induced at the beginning of the sexual cycle and produces acety l-CoA, which most probably is a prerequisite for fruiting body formation du ring later stages of sexual development, We discuss the contribution of ACL activity to the life cycle of S. macrospora.