Isolation and characterization of the amino-acid pools located within the cytoplasm and vacuoles of Candida utilis

Abstract

Two distinct amino-acid pools were demonstrated in the food yeast Candida utilis. Treatment of the cells with basic protein (cytochrome c) under isotonic conditions permeabilized the plasmalemma but left the tonoplast intact. The selective effect on these membranes was indicated by the observation of intact vacuoles but changed contrast of the cytoplasm in the phase-contrast microscope and by the free access of a chromogenic substrate to a cytoplasmic enzyme (α-glucosidase). However, only 10-20% of the soluble amino acids were released from the cells and these had a rapid turnover as demonstrated by pulse labelling experiments using (14)C(U)-arginine, (14)C(U)-glucose, and (15)N-ammonia. This indicates a rapidly metabolized amino-acid pool located within the cytoplasm. Osmotic shock with water following the treatment with basic protein disrupted the tonoplast, an event which could be followed by phase-contrast microscopy. Most of the remaining amino acids were then released. These showed a slow turnover in pulse-labelling experiments and a high proportion of basic, nitrogen-rich amino acids, indicative of a storage function. The significance of such vacuolar and cytoplasmic pools in the regulation of cellular metabolism is discussed.