Aminopeptidase yscII of yeast

Abstract

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.

Molecular cloning of soluble aminopeptidases from Saccharomyces cerevisiae

“…Colonies or patches of yeast cells were examined for aminopeptidase activity by using a modification of the procedure described by Hirsch et al [24]. Cells grown on MV agar were overlaid with 15 ml of a solution containing 0.9% agar, 90 mM sodium phosphate pH 6.5, 1 mM Leu-NH-Nap in dimethyl sulfoxide and 8.25 mg fast garnet GBC.…”

“…The stacking gel was 4.0%, the separating gel was 7.S0/o. Aminopeptidase activity staining was carried out as described [24].…”

“…Analysis of a mutant with a thermosensitive aminopeptidase yscI1 activity, as well as gene dosage studies, provided evidence that APE2 is the structural gene of the enzyme [24]. No vital consequences of aminopeptidase yscll deficiency on cell life could be detected, but the enzyme was shown to be involved in the cellular supply of leucine from externally offered leucine-containing dipeptide substrates [24].…”

“…Analysis of a mutant with a thermosensitive aminopeptidase yscI1 activity, as well as gene dosage studies, provided evidence that APE2 is the structural gene of the enzyme [24]. No vital consequences of aminopeptidase yscll deficiency on cell life could be detected, but the enzyme was shown to be involved in the cellular supply of leucine from externally offered leucine-containing dipeptide substrates [24]. To gain further insight into the structure, function and localization of yeast soluble non-vacuolar aminopeptidases, plasmids containing genes responsible for aminopeptidase yscll, yscIII and yscIV activities were identified and the APE2 gene encoding aminopeptidase yscII was sequenced .…”

“…In a first attempt to analyze the function of aminopeptidase yscII, mutants devoid of this activity have been isolated and characterized [24]. Analysis of a mutant with a thermosensitive aminopeptidase yscI1 activity, as well as gene dosage studies, provided evidence that APE2 is the structural gene of the enzyme [24].…”