Cellular compartmentation of aromatic amino acids in Neurospora crassa. II. Synthesis and misplaced accumulation of phenylalanine in Phen-2 auxotrophs

Abstract

Two mutants which require phenylalanine for normal growth and which show no prephenate dehydratase activity in vitro have been found to accumulate and excrete phenylalanine when incubated on minimal medium or grown on low concentrations of phenylalanine. The high levels of phenylalanine accumulated in these mutants apparently cannot be used for protein synthesis or for the regulation of the biosynthetic enzymes in the aromatic pathway. Mutant mycelia grown in high phenylalanine maintain a much lower level of free phenylalanine in the cells than do those grown on low phenylalanine or those which eventually grow on minimal. If all the phenylalanine required for the protein in a 3-day mycelial pad is supplied, little or no phenylalanine can be found in the medium after 3 days: if only a fraction of the total protein phenylalanine is supplied, the concentration of phenylalanine in the medium after 3 days is actually higher than the initial concentration. It is proposed that the mutation in these organisms has resulted in abnormal compartmentation of the phenylalanine produced so it cannot be utilized by the cells until it has been excreted and transported back into the normal pool channels. In this case, the transport (exogenous) and protein synthesis pools would be involved. The abnormal mislocation of the phenylalanine in the cell might be a result of the diffusion of free prephenate to low pH regions in the cell where it is nonenzymatically converted to phenylpyruvate. If, however, the mutant prephenate dehydratase is active in vivo, the mutation must somehow affect the activity or stability of the enzyme in vitro and also cause the release of the end product in the wrong place in the cell. This might be expected if the normal wild-type prephenate dehydratase is directionally oriented, e.g., as a result of membrane association, to release the product into normal cell channels (protein synthesis pool) while such oriented release might not occur in the mutants.