External and Internal Forms of Yeast Aminopeptidase II

Abstract

1. Intact cells of Succhuromyces cerevisiue catalyze the hydrolysis of various aminopeptidase substrates. This activity is not due to permeation of substrates and products but exerted by an external enzyme.2. From its substrate specificity and the effects of pH and inhibitors the enzyme was identified as aminopeptidase 11.3. About 40 ' %; of total aminopeptidase I1 activity is detectable with untreated exponentially growing cells. Up to two thirds of the external enzyme is released into the medium during enzymic digestion of the cell wall, while little enzyme is liberated by osmotic shock. Membrane preparations contained only small amounts of aminopeptidase I1 ; thus, the localization of the external enzyme appears to be similar to that of the so-called 'periplasmic' yeast hydrolases.4. By cytochemical methods the presence of aminopeptidase I1 in the cell envelope was visualized.5. In contrast to aminopeptidase 11, yeast dipeptidase is an entirely intracellular enzyme.The proteolytic system of yeasts has attracted considerable attention and is still most actively studied. Saccharomjws cerevisiue contains two proteinases and at least one enzyme with carboxypeptidase specificity [1,2]. In addition, three aminopeptidases and a single dipeptidase were found in yeast [3]. Aminopeptidase I, the proteinases and carboxypeptidase Y are enriched in vacuoles, and recent results suggest that these enzymes are regulated in a concerted way [3]. It remained unclear, however, in which way aminopeptidase 11, the other main aminopeptidase activity of yeast, and dipeptidase are distributed intracellularly. Preliminary experiments indicated that an extracellular aminopeptidase activity described earlier [4] could be brought about by aminopeptidase 11. We report here the'results of further studies aimed to establish the localization and catalytic properties of the external enzyme and, if possible, to demonstrate its identity with one of the known peptidases. MATERIALS AND METHODS Yeast Strain and Cultivation Conditions

Autolytic Release of Mannoproteins from Cell Walls of Saccharomyces cerevisiae

“…To date, only two proteases (aminopeptidases) are thought to be localized outside the yeast cytoplasmic membrane (Frey & Rohm, 1979;Trumbly & Bradley, 1983). The relationship between these proteases and those whose existence is suggested from our results remains to be elucidated.…”

Molecular cloning of soluble aminopeptidases from Saccharomyces cerevisiae

“…Large-scale soluble extracts were prepared as described by Suirez-Rendueles et al [42]. Aminopeptidase activity in soluble extracts against Leu-NH-Np was determined in 0.1 M Tris/HCl pH 7.5 according to [21]. The liberated 4-nitroaniline was measured in a Shimazdu spectrophotometer at 405 nm and activity was calculated using a molar absorption coefficient of 9500 1 x mol-x cm-'.…”

“…Plasmids purified from bacteria and reintroduced into yeast strain [11][12][13][14][15][16][17][18][19][20][21] in each case conferred the anticipated phenotype, thus demonstrating that the insert encoded a gene responsible for the restored aminopeptidase activity. Restriction analysis of the plasmids allowed them all to be classified into three groups (PAL, pGA and pNA), each group containing plasmids confering a distinct aminopeptidase activity band (Fig.…”

“…Only the high-molecular-mass vacuolar aminopeptidase yscI (also called LAP IV, for nomenclature see [19]), has been unambigously characterized [7, 81. Aminopeptidase yscII (also called LAP I) accounts for most of the cellular aminopeptidase activity in exponentially growing cells [20]. In strains isolated from commercial brewer's yeast, about half of the enzyme is found in the periplasmic compartment, the rest being intracellular [21]. Purification of the enzyme starting from yeast homogenates led to the isolation of two molecular forms which were catalytically indistinguishable but differed in molecular mass (100 and 140 kDa, respectively) [22].…”

The Cellular Location of Proteases in Candida albicans

“…cerevisiae (Frey & Rohm, 1979). The rates of hydrolysis (nmolmin-l ml-l) of a series of Pnaphthylamide substrates (140 p~) were : Leu-Nna, 15 ; Pro-Nna, 13 ; Arg-Nna, 13 ; Ala-Nna, 11 ; Phe-Nna, 9; Leu-4-methoxy-Nna, 6 ; Met-Nna, 5.…”