Characterization of vacuolar arginine uptake and amino acid efflux in Neurospora crassa using cupric ion to permeabilize the plasma membrane

Treatment of Neurospora crassa mycelia with cupric ion has been shown to permeabilize the plasma and mitochondrial membranes. Permeabilized mycelia were shown to take up arginine into the vacuoles. Uptake was ATP-independent and appeared to be driven by an existing K+-gradient. The kinetic characteristics of the observed uptake were similar to those observed using vacuolar membrane vesicles: the K-m for arginine uptake was found to be 4.2-4.5 mM. Permeabilized mycelia were used to study the regulation of arginine uptake into vacuoles, The results suggest that uptake is relatively indifferent to the contents of the vacuoles and is not affected by growth of mycelia in amino acid-supplemented medium, Efflux of arginine, lysine, and ornithine from vacuoles was also measured using mycelia permeabilized with cupric ion. Arginine release was shown to be specifically enhanced by cytosolic ornithine and/or increases in the vacuolar pool of arginine or ornithine. Lysine efflux was shown be indifferent to the presence of other amino acids, These observations emphasize the importance of vacuolar compartmentation in controlling arginine and ornithine metabolism and suggest that vacuolar compartmentation may play an important role in nitrogen homeostasis of filamentous fungi. (C) 1997 Academic Press.