The vacuoles of Neurospora crassa, grown in minimal medium, contain a 1:1 ratio of basic amino acids and phosphate, the latter in the form of long-chain, inorganic polyphosphate-P. Vacuoles isolated from cells depleted of polyphosphate retain basic amino acids despite the absence of over 90% of their polyphosphate. Thus, vacuolar retention of basic amino acids is not dependent upon binding to or charge neutralization by polyphosphate. Polyphosphate was found to be the only macromolecular polyanion in vacuoles of normal or phosphate-depleted cells. Gel filtration experiments revealed that about half the polyphosphate of normal vacuoles is bound strongly by vacuolar spermidine, Mg2+, and Ca2+. The polyphosphate thus occupied was not available for basic amino acid binding. We have identified about 90% of the cations of isolated vacuoles; in addition to spermidine, Mg2+, and Ca2+, the cation pool consists mainly of arginine, ornithine, histidine, lysine, and Na+, with a small amount of K+. Isolated vacuoles appear to be almost wholly impermeable to all these ions, and in vivo, vacuoles appear to be highly selective in ion uptake by an active process. The interaction of basic amino acid with the available polyphosphate was found to reduce the chemical activity of the former. In keeping with this effect, cells with abnormally high basic amino acid-polyphosphate ratios displayed greatly swollen vacuoles, indicating considerable osmotic activity of the basic amino acids and their counterions under these conditions.

A replica-printing technique has been developed for permeabilized colonies of Neurospora and yeast by which endogenous amino acid pools are visualized. By the use of cation-exchange paper, the method has detected Neurospora mutants unable to store the large pools of basic amino acids characteristic of wild type. Mutants of this sort were sought to study the function of the fungal vacuole.

The polyamines putrescine, spermidine, and spermine are multivalent cations that bind to anionic cell constituents such as nucleic acids. Their distribution between free and bound states within the cell is not known. Such knowledge would be important in relation to the negative control of polyamine synthesis. We report a tracer experiment in which [14C]ornithine was added to logarithmically growing Neurospora crassa mycelia. The amount and the specific radioactivity of the three polyamines thereafter suggested that new molecules of spermidine were made preferentially from new molecules of putrescine, and that new molecules of spermine were made from new molecules of spermidine. The extent of mixing of new [14C]- and resident [12C]spermidine indicated that 70% or more of the resident spermidine was sequestered, and not immediately accessible to spermine synthase. Cell fractionation revealed that about 28% of the cellular spermidine was vacuolar, and nonexchangeable with [14C] spermidine added at the time of cell breakage. We suggest that the remainder of sequestered spermidine is bound strongly to anionic sites in the cell, and is relatively inactive in the control and synthesis of polyamines.

At least 78%, and perhaps all, of inorganic polyphosphate is shown to be contained within the vesicles (vacuoles) of Neurospora crassa, where over 97% of the soluble arginine, lysine, and ornithine pools are known to accumulate. Furthermore, synthetic polyphosphate can concentrate arginine up to 400-fold from dilute (0.01 mM) solutions in equilibrium dialysis. For these reasons and because the molar ratio of basic amino acids and polyphosphate phosphorus is approximately 1, we tested the hypothesis that there was an obligate physiological relationship between them. Experiments in which nitrogen starvation and arginine excess were imposed upon cells showed that polyphosphate content was insensitive to changes in the basic amino acid content. Experiments involving phosphate starvation and restoration showed that basic amino acid content was almost wholly independent of polyphosphate pools. Moreover, the normal high degree of compartmentation of arginine in vesicles was maintained despite polyphosphate depletion, and arginine was still exchanged across the vesicular membrane. We conclude that N. crassa, like yeasts, can regulate polyphosphates and basic amino acids independently, and that the accumulation of basic amino acids in vesicles may depend upon an energy-requiring mechanism in addition to the demonstrated charge interaction with polyphosphate.