Compartmentation of the Tryptophan‐Synthase‐Proteolyzing System in Saccharomyces cerevisiae

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Abstract
Ficoll‐gradient fractionation of a metabolic lysate from yeast spheroplasts was used to prepare vacuole, mitochondria and cytosol fractions. Tryptophan synthase activity was exclusively found in the soluble fraction. Proteinases A, B and C as well as the tryptophan‐synthase‐inactivating activity were mainly found in the vacuole fraction. The heat‐stable activities inhibiting pröteinase and tryptophan‐synthase‐inactivating enzyme appeared in the soluble fraction. The subcellular separation of tryptophan synthase and the tryptophan‐synthase‐inactivating activities explains why in crude extracts from stationary yeast cells, high tryptophan synthase activities are observed in spite of high tryptophan‐synthase‐inactivating activity. The proteinases of a freshly prepared vacuole fraction from yeast spheroplasts are unable to inactivate externally added tryptophan synthase unless the organelles are disrupted by sonication or osmotic shock.