Regulation of the yeast vacuolar aminopeptidase Y gene (APY1) expression.

The steady-state levels of the aminopeptidase Y (APY1) transcript and the level of assayable aminopeptidase Y activity were measured under a variety of nutritional conditions. Specific APY1 mRNA is less abundant in cells grown in minimal medium than in cells grown in rich medium, while active enzyme levels follow just the opposite pattern. Aminopeptidase Y activity decreases when cells are deprived of glucose without a concomitant fall in mRNA levels. Production of aminopeptidase Y is not markedly affected by nitrogen regulation. APY1 gene expression is not disturbed in heat-shocked cells. Our data support the idea that the main control event in vacuolar aminopeptidase Y expression is a post-transcriptional step.

Identification of regulatory proteins that might be involved in carbon catabolite repression of the aminopeptidase I gene of the yeast Saccharomyces cerevisiae.

Transcription of the vacuolar aminopeptidase yscI (APE1) gene in Saccharomyces cerevisiae has previously been suggested to require the participation of a cis upstream activation sequence (UAS) involved in carbon catabolite repression that responds to glucose. To determine the structure of the APE1 UAS element, we used the 18-bp sequence 5'-ATGAATTAGTCAGCTTCT-3' as the DNA-binding site. Using gel mobility shift assays, we have identified a 78 kDa protein from yeast that binds specifically to both single and double-stranded forms of the UAS DNA-binding site. We have also identified a 48 kDa heterodimer from yeast that binds specifically to the single-stranded form of the UAS and whose DNA binding activity is remarkably heat stable. Even though the APE1 UAS contains a consensus sequence for the binding of the yeast activator protein yAP1, the two DNA-protein complexes could still be detected in a strain bearing a deletion in the YAP1 gene.

Transcriptional regulation of the yeast vacuolar aminopeptidase yscI encoding gene (APE1) by carbon sources.

Transcription of the vacuolar aminopeptidase yscI-encoding gene (APE1) is regulated by the carbon source used for yeast growth, responding to carbon catabolite repression. By Northern blot analyses, we determined the kinetics of glucose repression in growth-shift experiments. When added to induced cells, glucose leads to the disappearance of hybridizable aminopeptidase yscI RNA sequences within 30 min. However, the amount of inmunoreactive protein, once induced, is not affected by the addition of glucose. By deletion analysis of the fusion gene APE1-lacZ we have identified a number of strong regulatory regions in the APE1 promoter. Consensus sequences for the binding of yAP1 and the HAP2/HAP3/HAP4 complex are contained in those regions. Control of the APE1 gene expression is not mediated by the HXK2 regulatory gene, but a strain bearing a deletion in the CAT1 gene can not derepress APE1 transcription to wild-type levels.

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated, sorted and matured in the fission yeast Schizosaccharomyces pombe.

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPYsc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 mu derived plasmid. Immunoblot analysis revealed that CPYsc produced in the fission yeast has a higher molecular mass than mature CPYsc produced by the budding yeast. CPYsc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPYsc produced by S. cerevisiae. CPYsc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPYsc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.

Cis and trans-acting regulatory elements required for regulation of the CPS1 gene in Saccharomyces cerevisiae.

To clarify the transcriptional regulation by nutrient limitation of the gene encoding carboxypeptidase yscS in Saccharomyces cerevisiae (CPS1), we performed an analysis of its 5' noncoding region. In deletion experiments a sequence located between positions -644 and -591 was found to be responsible for transcriptional repression of the CPS1 gene in yeast cells grown on rich nitrogen sources. Furthermore, a 162 bp fragment spanning positions -644 to -482 of the promoter of the CPS1 gene repressed gene expression when placed 3' to the upstream activation sequence (UAS) of the heterologous gene CYC1. A fragment containing this putative upstream repression sequence (URS) was shown specifically to bind protein from a yeast extract as demonstrated by gel retardation experiments. Although a sequence mediating the control of gene expression by GCN4 was found within the URS element, the GCN4 gene product is not required for DNA-binding activity. In addition, at least three other upstream activation UASs responsible for the activation of CPS1 expression by glucose under nitrogen starvation conditions were found to be located between positions -673 and -644, -482 and -353, and -243 and -186, respectively. The putative mechanism of the nitrogen limitation-dependent regulation of CPS1 expression via these regulatory elements is discussed.

Dipeptidyl aminopeptidase yspI mutants of Schizosaccharomyces pombe: genetic mapping of dpa1+ on chromosome III.

A mutant strain of Schizosaccharomyces pombe lacking dipeptidyl aminopeptidase yspI was isolated from a strain already defective in aminopeptidase activity by means of a staining technique with the chromogenic substrate ala-pro-4-methoxy-beta-naph-thylamide to screen colonies for the absence of the enzyme. The defect segregated 2+:2- in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Gene dosage experiments indicated that the mutation resides in the structural gene of dipeptidyl aminopeptidase yspI, dpa1+. The dpa1+ gene was located on chromosome III by using m-fluorophenylalanine-induced haploidization and mitotic analysis. dpa1 mutants did not show any obvious phenotype under a variety of conditions tested.

Purification and characterization of aminopeptidase yspI from Schizosaccharomyces pombe.

Aminopeptidase yspI was purified to apparent homogeneity from the fission yeast Schizosaccharomyces pombe. The molecular mass of the native enzyme was estimated to be 184 kDa by gel filtration chromatography. A value of 92 kDa was calculated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is thus a dimer with two identical subunits. Optimum pH for cleavage of synthetic aminoacyl-4-nitroanilides is 7.0. Mercury ions, EDTA and chloroquine were found to be potent inhibitors of aminopeptidase yspI activity. Substrate specificity studies indicate that the purified enzyme cleaves L-lysine-4-nitroanilide with high efficiency.

Control of Saccharomyces cerevisiae carboxypeptidase S (CPS1) gene expression under nutrient limitation.

Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients. Enzyme production is sensitive to nitrogen catabolite repression; i.e. the presence of ammonium ions maintains expression of the gene at a low level. Transfer of ammonium-glucose pre-grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells. Derepression of the gene by nitrogen limitation is cycloheximide-insensitive. Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions. Non-metabolizable sugar analogs (2-deoxyglucose, 6-methyl-glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy-dependent. Production of carboxypeptidase yscS also increases several-fold when ammonium-pregrown cells are transferred to media containing glucose and a non-readily metabolizable nitrogen source such as proline, leucine, valine or leucyl-glycine. Analysis of CPS1 expression in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23 degrees C) and in heat-shocked cells (38 degrees C) shows that steady-state levels of CPS1 mRNA are not controlled by a low cAMP level-signalling pathway.

Purification and characterization of the endogenous inhibitor for proteinase B from Schizosaccharomyces pombe.

A rapid purification procedure for the endogenous inhibitor of proteinase yspB from Schizosaccharomyces pombe is described. Starting from a boiled extract, the purification procedure included an ionic exchange chromatography and two reverse phase chromatographies using a HPLC system. The molecular mass of the purified polypeptide was estimated to be 8,100 Da by gel filtration. The isoelectric point of the inhibitor was found to be 5.3 after electrofocusing of a purified preparation. The amino acid composition of the proteinase yspB inhibitor was analyzed after acid hydrolysis. The calculated number of residues was 67 and the corresponding molecular mass 7370 Da. There are several differences in the molecular characteristics between the inhibitor from Schizosaccharomyces pombe and the corresponding inhibitor previously purified from Saccharomyces cerevisiae which might reflect the evolutionary divergence between the two yeast genera.

Molecular cloning of soluble aminopeptidases from Saccharomyces cerevisiae. Sequence analysis of aminopeptidase yscII, a putative zinc-metallopeptidase.

Plasmids capable of complementing lap1, lap2 and lap3 mutations [R.J. Trumbly and G. Bradley (1983) J. Bacteriol. 156, 36-48] were isolated from a yeast YEp13 library by screening for activity against the chromogenic aminopeptidase substrate L-leucine beta-naphthylamide in intact yeast colonies. The genomic inserts were shown to contain the structural genes for aminopeptidases yscII, yscIII and yscIV. Plasmids containing the gene encoding aminopeptidase yscII of Saccharomyces cerevisiae, APE2 (LAP1) were analyzed in detail. APE2 was determined by DNA blot analysis to be a single-copy gene located on chromosome XI. The cloned fragment was used to identify a 2.7-kb mRNA. The cloned APE2 gene was sequenced and found to consist of an open reading frame of 2583 bp encoding a protein of 861 amino acids. The protein sequence contains two putative N-glycosylation sites. A significant amino acid similarity was detected between the APE2 gene product and members of the zinc-dependent metallopeptidase gene family. Chromosomal disruption of the APE2 gene completely abolishes the distinct activity band previously identified as aminopeptidase yscII [H.H. Hirsch, P. Suárez-Rendueles, T. Achstetter and D.H. Wolf (1988) Eur. J. Biochem. 173, 589-598] in crude extracts subjected to non-denaturing polyacrylamide gel electrophoresis and subsequent aminopeptidase activity staining. No vital consequence of aminopeptidase yscII absence on cell growth could be detected.

Isolation and characterization of Schizosaccharomyces pombe mutants lacking aminopeptidase activity.

A mutant strain of the fission yeast Schizosaccharomyces pombe defective in aminopeptidase I was isolated by screening for lack of activity against the chromogenic substrate lysine-beta-naphthylamide in isolated colonies. 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 chromosomal gene mutation. Gene dosage experiments indicated that the mutation might reside in the structural gene of aminopeptidase I. No vital consequences of aminopeptidase I deficiency on cell life and sporulation could be detected. However, the enzyme seems to be involved in protein degradation under conditions of nutrient deprivation.

The proteolytic system of the fission yeast Schizosaccharomyces pombe.

Proteinase and peptidase activities of the fission yeast Schizosaccharomyces pombe were investigated. Several intracellular proteolytic enzymes were found: two endoproteinases, one carboxypeptidase, one aminopeptidase and one dipeptidyl-aminopeptidase. In addition, proteinase inhibitors were detected. In fresh crude extracts an activation procedure is needed to measure maximal activities of endoproteinases and carboxypeptidase, whose level is markedly dependent on growth medium composition and on growth phase, while aminopeptidase and dipeptidyl-aminopeptidase activities are very little, if at all, regulated by the carbon source.

Molecular cloning and sequencing of genomic DNA encoding yeast vacuolar carboxypeptidase yscS.

A Saccharomyces cerevisiae genomic DNA encoding vacuolar carboxypeptidase yscS was cloned from a yeast YEp13 library by complementation of the previously characterized mutation cps1-1 [(1981) J. Bacteriol. 147, 418-426], by means of staining carboxypeptidase activity in yeast colonies. The nucleotide sequence of the cloned gene was determined. The open reading frame of CPS1 consists of 576 codons and therefore encodes a protein of 64961 molecular weight. A stretch of 19 residues near the N-terminus of the deduced polypeptide sequence contains characteristics common to known hydrophobic leader sequences. CPS1 was determined by DNA blot analysis to be a single copy gene located on chromosome X. The cloned fragment was used to identify a 2.1 kb mRNA. A transcriptional activation of CPS1 occurs when cells grow on a substrate of carboxy-peptidase yscS as sole nitrogen source.

The proteinase yscB inhibitor (PB12) gene of yeast and studies on the function of its protein product.

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, 12,555-12,561] 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 URA3 gene 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.

Biochemical and genetic analysis of an alpha-mannosidase mutant from Saccharomyces cerevisiae.

A yeast mutant lacking non-specific alpha-mannosidase activity was found as a background marker during our search for dap2 mutants (Suárez-Rendueles, P. and Wolf, D. H. (1987) J. Bacteriol. 169, 4041-4048). The mutant (DPS-15) is characterized in detail. The mutation called amd1 segregates 2:2 in meiotic tetrads, indicating a single chromosomal gene mutation which is recessive. Diploids heterozygous for amd1 show gene dosage. Thus, it appears that AMD1 might be the structural gene for alpha-mannosidase. Results obtained with this mutant show that alpha-mannosidase is not a vital component of the vegetative cell cycle. The differentiation process of sporulation is not disturbed in homozygous mutant diploids. Mannose turnover does not seem to be altered in mutant cells.

Localization of dipeptidyl aminopeptidase yscIV in the plasma membrane of Saccharomyces cerevisiae.

The subcellular distribution of dipeptidyl aminopeptidase activity was studied in protoplast lysates of Saccharomyces cerevisiae that were virtually free from vacuolar contamination. Dipeptidyl aminopeptidase yscIV, the STE13 gene product, was found to be associated with plasma membrane vesicles after sucrose gradient isopycnic centrifugation. Another dipeptidyl aminopeptidase activity, not yet fully characterized, was localized in a microvesicular population co-sedimenting with chitosomes.

Yeast vacuolar aminopeptidase yscI. Isolation and regulation of the APE1 (LAP4) structural gene.

The structural gene, APE1, (LAP4), for the vacuolar aminopeptidase I of Saccharomyces cerevisiae was cloned with the aid of a staining technique which permitted monitoring of aminopeptidase activity in yeast colonies. Genetic linkage data demonstrate that integrated copies of the cloned gene map to the APE1 locus. The nucleotide sequence of the cloned gene was determined. The open reading frame of APE1 consists of 514 codons and, therefore, encodes a larger protein (MW 57,003) than the reported mature aminopeptidase yscI (MW 44,800), suggesting that proteolytic processing must occur. A 1.75-kb mRNA, which is made in substantial amounts only when yeast cells have exhausted the glucose supply, was identified.

Aminopeptidase yscII of yeast. Isolation of mutants and their biochemical and genetic analysis.

Mutant strains of the yeast Saccharomyces cerevisiae defective in aminopeptidase yscII were isolated by screening for reduced external activity against the chromogenic substrate lysine beta-naphthylamide. One of the selected mutant strains analyzed in detail showed wild-type staining activity when tested at 23 degrees C but mutant activity after exposure to 37 degrees 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 beta-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.

Identification of the structural gene for dipeptidyl aminopeptidase yscV (DAP2) of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae lacking dipeptidyl aminopeptidase yscV were isolated from a strain already defective in dipeptidyl aminopeptidase yscIV, an enzyme with overlapping substrate specificity. The mutants were identified by a staining technique with the chromogenic substrate Ala-Pro-4-methoxy-beta-naphthylamide to screen colonies for the absence of the enzyme. One of the mutants had a thermolabile activity, indicating that it contained a structural gene mutation. The 53 mutants analyzed fell into one complementation group that corresponded to the yscV structural gene, DAP2. The defect segregated 2:2 in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Diploids heterozygous for DAP2 displayed gene dosage effects with respect to yscV enzyme activity. The absence of dipeptidyl aminopeptidase yscV or the combined loss of both dipeptidyl aminopeptidases yscIV and yscV did not affect mitotic growth under rich or poor growth conditions. In contrast to the dipeptidyl aminopeptidase yscIV lesion (ste13), which leads to alpha sterility because strains secrete incompletely processed forms of the alpha-factor pheromone, the dipeptidyl aminopeptidase yscV lesion did not affect mating, and strains produced fully active alpha-factor pheromone. dap2 mutants did not show any obvious phenotype under a variety of conditions tested.