Lack of mitochondrial citrate synthase discloses a new meiotic checkpoint in a strict aerobe

Abstract

Mitochondrial citrate synthase (mCS) is the initial enzyme of the tricarboxylic acid (TCA) cycle. Despite the key position of this protein in respiratory metabolism, very few studies have addressed the question of the effects of the absence of mCS in development. Here we report on the characterization of 15 point mutations and a complete deletion of the cit1 gene, which encodes mCS in the ®lamentous fungus Podospora anserina. This gene was identi®ed genetically through a systematic search for suppressors of the metabolic defect of the peroxisomal pex2 mutants. The cit1 mutant strains exhibit no visible vegetative defects. However, they display an unexpected developmental phenotype: in homozygous crosses, cit1 mutations impair meiosis progression beyond the diffuse stage, a key stage of meiotic prophase. Enzyme assays, immuno¯uorescence and western blotting experiments show that the presence of the mCS protein is more important for completion of meiosis than its wellknown enzyme activity. Combined with observations made in budding yeast, our data suggest that there is a general metabolic checkpoint at the diffuse stage in eukaryotes.

Peroxisomal ABC transporters and β-oxidation during the life cycle of the filamentous fungus Podospora anserina

“…NotI is located 30 bp downstream the Start codon and BbvCI 50 bp upstream the Stop codon of pABC2 (creating pUCDFDC2). The DNA fragment containing the hph gene was amplified by PCR from pUC-Hygro (Ruprich-Robert et al, 2002b). Two restriction sites were added at the primers extremity as follows: 5 0 -Not-CCAACTGGTTCCCGGTC-3 0 and -5 0 -BbvCI-CCCAACTGATATTGAAG G-3 0 allowing orientated cloning of the selective marker.…”

“…The open reading frames were replaced by the Escherichia coli hph gene (conferring hygromycin resistance). For fox2, 5 0 and 3 0 flanking sequences were amplified by PCR and inserted into pUC-Hygro (Ruprich-Robert et al, 2002b) vector on both sides of the hph gene. The 5 0 flanking sequence was amplified using Fox2promHIII (5 0 -GACAAGCTTAACCGAACCGAGTTGACATC-3 0 ) and Fox2promBI (5 0 -GACGGATCCGAGGAATGGTGGTGAAGGAA-3 0 ) primers, the underlined nucleotides corresponding, respectively, to the HindIII and BamHI restriction sites added for cloning this 558 pb fragment downstream of the hph gene.…”

Metabolic and Developmental Effects Resulting from Deletion of thecitAGene Encoding Citrate Synthase in Aspergillus nidulans

“…In P. anserina, deletion or lossof-function mutants of the citrate synthase gene cit1 can grow on standard medium that contains dextrin, presumably a limiting carbon source, and 25% of wild-type citrate synthase activity is present in the mutants. Furthermore, Western blot assays showed a second weaker cross-reacting band in extracts from all strains when a polyclonal antibody to S. cerevisiae Cit1 was used (38). This additional citrate synthase is likely to be methylcitrate synthase, and a predicted gene is found in the P. anserina genome (PODANSg3467; accession no.…”

“…Respiration mutants are female sterile (13). The effects of mutations in the citrate synthase gene, cit1, on sexual development in the heterothallic fungus P. anserina have been subject to detailed study (38). Immunofluorescence studies demonstrated the presence of mitochondrial localized protein in asci and ascospores.…”

Meiosis in Mycelial Fungi

“…Also, very little is known concerning the metabolic requirements ensuring correct meiotic and/or sporulation progression. For example, why do P. anserina mutants defective for the mitochondrial citrate synthase arrest at the diffuse stage, a major landmark of oogenesis (Ruprich-Robert et al 2002)? In short, the discovery of new proteins involved in the meiotic process often simultaneously provides satisfying answers to long-standing questions in chromosome biology, while revealing a new set of puzzles to solve.…”