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Strain Genotype Source or reference MATa MATot stel3-1 ade6 leul trpS his6 met) can) rmel gal2 MATa sstl-2 ade2 ural his6 metl cyh2 rme MATa sst2-1 ade2 ural his6 met) cyh2 rme MATa pral-J prbl-l prcl-) cpsl-3 ade2 his MATot ste)3-1 prbl-l prcl-) cpsl-3 ade2 his MATot stel3-1 prbl-l prcl-l cpsl-3 ape2-1 leu2 his MATa prcl-l cpsl-3 ape2-1 leu2 MATa prcl-l cpsl-3 ape2-1 leu2 lys2 MATa stel3-1 dap2-1 prbl-l prcl-J cpsl-3 ape2-1 leu2 his MATa stel3-1 dap2-2 ade6 leul trpS his6 met) can) rmei gaI2 MATa stel3-1 dap2-1 prbl-) prcl-i cpsl-3 ape2-1 leu2 Iys2 his MATa stel3-1 dap2-1 prcl-l cpsl-3 ape2-1 leu2 lys2 MATa stel3-1 prb)-J prcl-) cpsl-3 ape2-1 leu2 MATas dap2-1 prcl-l cpsl-3 ape2-1 leu2 his MATa stel3-1 dap2-1 prbl-) prcl-l cpsl-3 ape2-1 leu2 lys2 his MATa stel3-1 dap2-1 prcl-l cpsl-3 ape2-1 leu2 lys2 his MATxt stel3-1 prcl-l cpsl-3 ape2-1 leu2 lys2 his MATat dap2-1 leu2 his MATa dap2-1 prcl-J cpsl-3 ape2-1 his MATot dap2-1 prcl-l cps)-3 ape2-1 his MATa stel3-1 dap2-1 prcl-1 cpsl-3 ape2-1 ade6 leu his MATa stel3-1 prcl-l cps)-3 ape2-1 ade6 leu his MATa stel3-1 dap2-2 prcl-) met) MATot stel3-1 dap2-2 prbl-l cpsl-3 trp5 MATa stel3-1 dap2-2 prbl-l prcl-) trp5 leu met) MATa steB3-1 dap2-2 prbl-I ade6 metl MATa stel3-1 cpsl-3 trp5 his6 ade6 leu MATot stel3-1 prc)-) cpsl-3 his6 MATa stel3-1 prcl-l ade6 leu met) MATot stel3-1 prbl-l prc)-) cpsl-3 trpS his6 leu Yeast Genetic Stock Center 29 9 9 B. Mechler and D. H. Wolf, unpublished

Mutant strains of the yeast Saccharomyces cerevisiae defective in aminopeptidase yscII were isolated by screening for reduced external activity against the chromogenic substrate lysine β‐naphthylamide. One of the selected mutant strains analyzed in detail showed wild‐type staining activity when tested at 23°C but mutant activity after exposure to 37°C, suggesting a temperature‐sensitive mutation. Electrophoretic separation of mutant crude extracts on non‐denaturing polyacrylamide gels and subsequent activity staining using lysine and leucine β‐naphthylamides as substrates revealed that in all strains isolated the same distinct activity band was affected, which corresponded to the aminopeptidase activity identified previously as aminopeptidase yscII [Achstetter, T., Ehmann, C. & Wolf, D. H. (1983) Arch. Biochem. Biophys. 226, 292–305]. All mutants strains isolated fell into the same complementation group. Tetrad dissection of sporulated diploids heterozygous for the wild‐type and mutant allele resulted in a 2:2 segregation of mutant and wild‐type phenotype indicating a single gene mutation. The characteristics of the mutations analyzed point to the gene which we called APE2 as the structural gene of aminopeptidase yscII. No vital consequences of aminopeptidase yscII deficiency on cell life and differentiation could be detected. However, the enzyme seems to be involved in the cellular supply of leucine from externally offered leucine‐containing dipeptide substrates.

The gene for proteinase yscB inhibitor I2B (PBI2) from Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library, and was sequenced. The gene codes for a single protein of 75 amino acids. In contrast to the published amino acid sequence [Maier, K., Müller, H., Tesch, R., Trolp, T., Witt, I. & Holzer, H. (1979) J. Biol. Chem. 254, 12555–12561] the DNA sequence revealed a valine instead of a leucine at position 33 (32 of the mature protein). Therefore the primary sequences of the isoinhibitors I2B of S. cerevisiae and I1B of Saccharomyces carlsbergensis differ only at position 34 (glutamic acid/lysine). The open reading frame of PBI2 was replaced in vitro by the URA3gene and a I2B null mutant of S. cerevisiae was constructed by gene replacement. The mutation resulted in an elevation of the protein degradation rate by 50% when grown under nutritional stress compared to the isogenic wild type. Growth and viability of the cells was not significantly affected by the absence of I2B.

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