Energy requirement for the mobilization of vacuolar arginine in Neurospora crassa

Abstract

The bulk of the intracellular arginine pool in exponentially growing mycelia of Neurospora crassa is sequestered in the vacuoles. Vacuolar arginine effluxes from the vacuoles into the cytosol and is catabolized to ornithine and urea upon nitrogen starvation. The energy requirement for mobilization has been studied by treating nitrogen-starved mycelia with inhibitors of respiration or glycolysis or an uncoupler of respiration. Mobilization was inhibited by the inhibitors or the uncoupler of respiration, but not by the inhibitors ofglycolysis. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge of the treated mycelia. The inhibitors of glycolysis reduced the ATP pool but had no effect on the energy charge. The results indicate that mobilization of arginine from the vacuoles requires metabolic energy. The forms of this energy and the mode of its associations with the mobilization process are discussed.Nutritional stresses stimulate a variety of metabolic processes which do not occur in organisms under normal growth conditions. In fungi, nitrogen starvation results in the expression of a number of metabolic systems which utilize alternative sources of nitrogen-containing metabolites. These systems include a variety of pathways leading to the production of ammonia (5) and transport systems for nitrogen-containing metabolites (7). All of these systems are repressed or impaired when the fungi are growing on a preferred nitrogen source such as ammonia. This phenomenon is called nitrogen metabolite control (7).One example of a nitrogen metabolite-regulated system is the catabolism of vacuolar arginine in response to nitrogen starvation in Neurospora crassa. Mycelia of this ascomycete have a large intracellular arginine pool. The bulk of this pool, >98%, is sequestered in the vacuoles (9,11). When mycelia are deprived of nitrogen, vacuolar arginine effluxes across the tonoplast into the cytosol, where it is catabolized as an alternative source of nitrogen. This phenomenon has been termed mobilization of vacuolar arginine (5). The mechanism of translocation of arginine across the tonoplast is not clear. It is not known whether this translocation involves an active transport mechanism regulated by nitrogen metabolite control or a facilitated exchange in which some metabolite(s) replaces or displaces arginine from the vacuole. There are indications that the level of intracellular glutamine might be involved in the mobilization of the vacuolar arginine (T. L. Legerton and R. L. Weiss, manuscript in preparation); glutamine appears to be the effector for the control of a number of nitrogen metabolite-regulated systems (7). This report deals with the energy requirement for mobilization. The effect of various energy inhibitors on mobilization of vacuolar arginine has been examined. Inhibitors of oxidative phosphorylation have been shown to inhibit mobilization. The data support the hypothesis that energy is necessary for the proper functioning of the mechanism that mobilizes vacuolar arginine...

Compartmental and regulatory mechanisms in the arginine pathways of Neurospora crassa and Saccharomyces cerevisiae.

“…A second finding was that inhibitors of glycolysis, which lowered the ATP pool but did not lower the energy charge, led to loss of arginine from the vacuole in vivo, even though glutamine was not seriously depleted. The loss was again blocked by respiratory inhibitors (75). The glycolytic derivative acting as a signal for retention or release of vacuolar arginine has not been identified.…”

“…The behavior of vacuolar arginine pools in living N. c(rassa mycelia is consistent with the energetics of vacuolar accumulation so far determined in vivo. Cells grown in minimal medium and labeled with [14C]arginine were treated with inhibitors of glycolysis, inhibitors or uncouplers of respiration, or both (74,75). Transport of cytosolic arginine into the vacuoles was blocked by inhibitors of respiration or uncouplers, which considerably lowered the energy charge.…”

“…This is consistent with the observation (42) that vacuoles retained arginine well in vitro (see above). Other manipulations showed that energy status is involved in mobilization of arginine from vacuoles (75). For instance, a glutamine deficiency is the metabolic signal denoting nitrogen starvation.…”

Significance of Copper in Alcohol Production with Fermentation of Raisin Extracts by the Cell Recycle Process

“…In each fermentation corresponding to a copper level, as well as those to study optimum glucose, a sample of yeast dry cells was collected after filtration, using Sartorius filter 0.45~~ washing and drying at room temperature. The dry biomass was added, in 5 mL toluene solution containing 4 g L-' PPO (2,5 diphenyloxazole) and 0.1 g.L-' POPOP [I,4 Bis [2-(5phenyl)-oxazolyl] benzol] (Drainas et al, 1982) 3000. The glucose uptake rate was calculated as nM glucose per min per mg dry weight Saccharomyces cerevisiae cells.…”

Isolation and Characterization of Vacuoles from the Ergot Fungus Claviceps purpurea

“…In the case of Neurospora and Saccharomyces species, vacuoles were found to contain high activities of hydrolytic enzymes and thus appear to be analogous to the lysosome of animal cells (13,15,20). The compartmentation of amino acids and inorganic polyphospates in vacuoles has been demonstrated (6,7,19). This paper deals with the isolation of vacuoles from two kinds of cells of C. purpurea that differ in their ability to synthesize ergot alkaloids.…”