The N-end rule of selective protein turnover: mechanistic aspects and functional implications.

2-fold. Lack of the vacuolar endoproteinases yscA and yscB reduces the protein degradation rate by about 30% under the conditions of canavanine-induced synthesis of false proteins (Table 2). The remaining protein degradation rate of 15% under starvation conditions, and of 6(r-70°h under growing conditions in mineral medium in strains defective in the two vacuolar endoproteinases yscA and yscB, clearly indicates that other degradative enzyme systems must exist in the yeast cell. This is also demonstrated by a strain carrying a false carboxypeptidase yscY protein owing to mutation in its structural gene (Wolf & Weiser, 1977) (Fig. 3). The false protein is degraded with a half-life of 30min in vivo in a strain with a wild-type proteinase background. Introduction of the proteinase yscA and proteinase yscB (as well as carboxypeptidase yscS) mutations into this strain does not alter the high degradation rate of the false caboxypeptidase yscY protein (Fig. 3). A multitude of new proteolytic enzymes has been found with the aid of chromogenic peptide substrates (Achstetter tv al., 1981, 1982, 1983, 1984a; Suarez-Rendueles et al., 1981; Wolf & Ehmann, 1981; Achstetter & Wolf, 19856). One new enzyme, identified as dipeptidyl aminopeptidase and called dipeptidyl aminopeptidase yscV is localized in the vacuolar membrance (Garcia-Alvarez et al., 1985). Mutants deficient in this enzyme activity do not show any altered phenotype, probably indicating that other enzymes with overlapping activity might exist which can substitute for dipeptidyl aminopeptidase yscV when it is missing (M. P. Suarez-Rendueles & D. H . Wolf, unpublished work). The majority of the new proteolytic enzymes are nonvacuolar (Emter & Wolf, 1984). Two proteinases, called proteinase yscD and proteinase yscE, were purified and characterized (Achstetter e t al., 19846, 1985). Mutants devoid of proteinase yscD were isolated, but no phenotype has been found as yet, probably indicating that other activity(ies) can substitute in vivo when proteinase yscD is absent (Garcia-Alvarez et a/., 1987). Proteinase yscE, an enzyme of M, > 600 000, has proven to be ATP stimulated (C. Escher & D. H . Wolf, unpublished work). Mutants of this enzyme, as well as the many other proteinases identified so far, should give insight into their function in vivo. Among the detected enzymes, additional proteinases exhibiting degradative capacity might be uncovered and proteinases of highly restricted and specific functions might be found. The best known example of this kind is proteinase yscF, the enzyme responsible for maturation of the a-factor precursor protein. The enzyme is highly specific for the recognition of sequences of two basic amino acids and splits the pheromone precursor at these recognition sites (Julius et a[., 1984; Achstetter & Wolf, 19856).