The proteinase yscA‐inhibitor, I^A₃, gene Studies of cytoplasmic proteinase inhibitor deficiency on yeast physiology

Abstract

i'h¢ gen¢ of" the proteina~ y~cA inhibitor i":j, PAl3, of the yeast Saceharan)yce,~ ¢¢ecvi~ta¢ was isolated by oligonucl¢otidc screening of a genomic DNA library and sequenced. The gent codes for a single protein of 68 amino acids, The structural PAI3 gen¢ was deleted in vitro by oli$onucleotidesite.directed mutagenests. The mutated allele was introduced via homologous recombination into the 8enome of wild,type yeast and into the Itenome of a yeast mutant, which lacks the s~ond cytoplasmic proteina~-inhibitor, l°a, The deficiency of either or of both inhibitors has no effect on th© cell viability under various physiological conditions, The inhibitor mutants, ho~vever, show an increase in the lleneral in vivo protein degradation rate, The 1^3 mutant has a 2-~l.fold increased protein degradation rate in the tint 6 h aft©r a shift from rich medium onto starvation-medium, whereas the IBz mutant shows a constantly incrca~d degradation rate of 20~50~ under the same conditions. The inhibitor double null mutant has the same protein degradation rate as the [^~ null mutant. The~ results suggest an in vivo interaction b~tw©en the vacuolor endopeptidases and their cytoplasmic inhibitors.Pt~nt¢olysis; Prot¢ina,~ inhibitor; [^~; [no; Null mutant; Saccharomycts cerevisia¢ 1, INTRODUCTIONThe vacuole (lysosome) of the yeast is an actdic compartment, which contains multiple hydrolases (see reviews [1][2][3][4]). It is also a storage organelle for several compounds like phosphate, amino acids and calcium. The budding yeast Saccharomyces cerevisiae contains 1-2 large vacuoles per cell, which undergo multiple fu. sion and fragmentation processes during cell growth and sporulation (for review, see [5]).The two vacuolar endopeptidases proteinase yscA and yscB are essential enzymes for protein degradation during vegetative growth and, most pronounced, under starvation conditions ([6], for review see [1][2][3][4]). Proteinase yscA is even vital for cell viability under starvetion conditions [6]. The peptidases proteinase yscA, proteinase yscB and earboxypeptidase yscY are necessary for the differentiation prbcess of sporulation [6].The cytoplasm of the yeast S:tccharomyces cerevisiae contains the peptide inhibitors, Ia3, In2 and I c, specific for the vacuolar endopeptida.,+es proteinase yscA, proteinase yscB and carbo×ypeptidase yscY, respectively [7,8] No proteinase activities can be detected in cell extracts of wild-type yeast due to the formation of the enzyme/inhibitor complexes. The proteinase act ivities can be recovered by incubation at pH 5 or in the presence of detergents [13][14][15][16]. The inhibitors were found in the cytoplasmic fraction and only traces were found in the microsomal fraction [7]. The proteinase inhibitors are small and heat stable proteins (IA~: 7.6 kDa; In2:8.5 kDa), which contain no disulphide bonds [17][18][19][20]. Little is known about the function of the cytoplasmic proteinase inhibitors.The intracellular distribution of the proteinases and their inhibitors, as well as the observation that they are ex...

The Potency and Specificity of the Interaction between the IA3 Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae

“…The recombinant protein that accumulated in E. coli was soluble and was purified to homogeneity from cell lysates by taking advantage of the His 6 tag introduced from the pET-22b vector at the C terminus of the IA 3 polypeptide chain (see "Experimental Procedures"). N-terminal analysis of one batch of the homogeneous wild-type inhibitor through 10 cycles of Edman degradation gave the sequence Asn-Thr-Asp-Gln-Gln-Lys-Val-Ser-Glu-Ile, which is exactly coincident with that predicted by the DNA sequence for residues 2-11 (Table I) (11) and indicates that the initiator Met 1 residue had been removed during the accumulation of this batch of recombinant protein in E. coli. Analysis of a separate batch of recombinant wild-type inhibitor by MALDI-TOF mass spectometry gave a mass of 8772 Da, identical with that predicted (8772 Da) for the IA 3 sequence plus the C-terminal extension of ϳLeu-Glu-His 6 introduced from the pET-vector.…”

“…The complete sequence of this 68-residue inhibitor has been elucidated (10,11) and the inhibitory activity of IA 3 has been shown to reside within the N-terminal half of the molecule (10,12). We have recently solved the structure of the IA 3 -proteinase A complex (12), demonstrating that whereas free IA 3 has little intrinsic secondary structure, residues 2-32 of the inhibitor, upon contact with proteinase A, become ordered and adopt a near perfect ␣-helical conformation occupying the active site cleft of the enzyme.…”

A Novel Function for the Cathepsin D Inhibitor in Tomato

“…In fact, the JIP21-deduced amino acid sequence presents an elevated identity with the family of CDIs described in potato (Mares et al, 1989;Ritonja et al, 1990;Strukelj et al, 1990;Hildmann et al, 1992;Maganja et al, 1992;Hannapel, 1993;Herbers et al, 1994;Ishikawa et al, 1994a;Kreft et al, 1997). Apart from this large family in potato, proteinaceous aspartic PINs are uncommon and are described in only a few plant species, yeast (Schu and Wolf, 1991), and the nematode Ascaris lumbricoides (AbuErreish and Peanasky, 1974). None of these inhibitors seems to be related to potato CDIs.…”

In silico Analysis and Characterization of a Putative Aspartic Proteinase Inhibitor, IA3 from Lachancea kluyveri

“…The IA3 inhibitor was first isolated and characterized from S. cerevisiae in 1974 [26]. The amino acid sequence of S. cerevisiae IA3 was then determined by using automated Edman degradation [27] and the gene coding for IA3 was successfully cloned and characterized [28]. The N-terminal half (residues 2-34) of the molecule has been shown to possess the inhibitory activity [25,28].…”

“…The amino acid sequence of S. cerevisiae IA3 was then determined by using automated Edman degradation [27] and the gene coding for IA3 was successfully cloned and characterized [28]. The N-terminal half (residues 2-34) of the molecule has been shown to possess the inhibitory activity [25,28]. Further evidence on the location of IA3 inhibitory activity was later revealed by Phylip et al [12].…”