FOLYLPOLYGLUTAMATE SYNTHESIS IN NEUROSPORA-CRASSA - TRANSFORMATION OFPOLYGLUTAMATE-DEFICIENT MUTANTS

The methionine auxotrophy of Neurospora crassa met-6 and mac mutants i s related to an inability to synthesize long-chained folylpolyglutamat es. Both of these lesions affect folylpolyglutamate synthetase activit y, but it is not clear whether these mutations occur in different gene s or in functional domains of the same gene. To address this question, copies of the met-6(+) gene have been introduced into both mutants us ing plasmid and cosmid vectors. Transformation to prototrophy was achi eved in both mutants. The ability of these mutant and transformant str ains to synthesize folylpolyglutamates was assessed by HPLC analysis o f folate cleavage products. Mycelial extracts of the wild type reveale d a folate pool dominated by folylhexaglutamates. These folates were a lso detected in the transformants but were lacking in both mutants. In the latter strains, the conjugated folates were mainly di- and triglu tamates. When incubated for 24 hr with [C-14]p-aminobenzoate, transfor mant and wild type cultures synthesized long chain folates, ca 60-80% of these being hexaglutamyl derivatives. In contrast, the labelled fol ates of mac and met-6 were mainly mono- and diglutamyl derivatives, re spectively. Polyglutamate synthesis was also studied in vitro by parti al purification and characterization of mycelial folylpolyglutamate sy nthetase protein. Mycelial extracts of the wild type and transformant cultures utilized 5,10-methylenetetrahydrofolate monoglutamate and its diglutamate as substrates in this synthetase reaction. In contrast, e xtracts of met-6 and mac mycelia utilized only one of these folate sub strates. Gel filtration of folylpolyglutamate synthetase protein indic ated apparent M(r) values of ca 66 000 in all strains. It is suggested that polyglutamate synthesis in Neurospora is probably mediated, as i n other eukaryotic species, by a single folylpolyglutamate synthetase protein.