ACTIVATION AND PROCESSING OF NONANCHORED YAPSIN-1 (YAP3P)

A C-terminally truncated form of yapsin 1 (yeast aspartic protease 3), the first member of the novel subclass of aspartic proteases with spe cificity for basic residues (designated the Yapsins), was overexpresse d and purified to apparent homogeneity, yielding similar to 1 mu g of yapsin 1/g of wet yeast, N-terminal amino acid analysis of the purifie d protein confirmed that the propeptide was absent and that the mature enzyme began at Ala(68). The mature enzyme was shown to be composed o f approximately equimolar amounts of two subunits, designated alpha an d beta, that were associated to each other by a disulfide bond. C-term inally truncated proyapsin 1 was also expressed in the baculovirus/Sf9 insect cell expression system and secreted as a zymogen that could be activated upon incubation at an acidic pH with an optimum at similar to 4.0, When expressed without its pro-region, it was localized intrac ellularly and lacked activity, indicating that the pro-region was requ ired for the correct folding of the enzyme, The activation of proyapsi n 1 in vitro exhibited linear kinetics and generated an intermediate f orm of yapsin 1 or pseudo-yapsin 1.