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

Abstract 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-β-naphthylamide 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, dpa 1⁺. The dpa 1⁺ gene was located on chromosome III by using l m- fluorophen-ylalanine-induced haploidization and mitotic analysis. dpa1 mutants did not show any obvious phenotype under a variety of conditions tested.     

Proteinase yscD mutants of yeast. Isolation and characterization

Mutants of the yeast Saccharomyces cerevisiae, devoid of proteinase yscD activity, were isolated by screening for the inability of mutagenized cells to hydrolyze Ac-Ala-Ala-Pro-Ala-beta-naphthylamide in situ. One of the selected mutants bears a thermolabile activity pointing to the gene called PRD1 as being the structural gene for proteinase yscD. All mutants isolated fell into one complementation group. The defect segregates 2:2 in meiotic tetrads indicating a single gene mutation, which was shown to be recessive. Diploids heterozygous for PRD1 display gene dosage. The absence of proteinase yscD did not affect mitotic growth under rich or poor growth conditions, neither mating nor ascopore formation. Also growth of mutant cells after a nutritional shift-down was not altered. Inactivation of enzymes tested which are subject to carbon-catabolite inactivation, a process proposed to be of proteolytic nature, is not affected by the absence of proteinase yscD. Protein degradation rates in growing cells, in cells under conditions of differentiation or heat shock, showed no obvious alteration in the absence of proteinase yscD activity. Also inactivation of alpha-factor pheromone was not affected by proteinase yscD absence. Normal growth of mutant cells on glycerol indicates that the enzyme is not involved in any vital event in mitochondrial biogenesis.     

Analysis and expression of STE13ca gene encoding a putative X-prolyl dipeptidyl aminopeptidase from Candida albicans

Candida albicans STE13ca gene was identified by its homology to the Saccharomyces cerevisiae STE13 gene that encodes for the dipeptidyl aminopeptidase A (DAP A) involved in the maturation of alpha-factor mating pheromone. Our study revealed that C. albicans ATCC 10231 depicts dipeptidyl aminopeptidase activity. We also analyzed the expression of the STE13ca gene homologue from this pathogenic yeast. This gene of 2793 pb is homozygotic and encodes for a predicted protein of 930 amino acids with a molecular weight of 107,035 Da. The predicted protein displays significant sequence similarity to S. cerevisiae Ste13p. This C. albicans gene is located in chromosome R. STE13ca gene increases its levels of expression in conditions of nutritional stress (proline as nitrogen source) and during formation of the germinal tube, suggesting a basic biological function for the STE13ca in this yeast.     

Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase

Alpha factor mating pheromone is a peptide of 13 amino acids secreted by Saccharomyces cerevisiae alpha cells. Nonmating ("sterile," or ste) alpha-cell mutants bearing defects in the STE13 gene do not produce normal alpha factor, but release a collection of incompletely processed forms (alpha factor) that have a markedly reduced specific biological activity. The major alpha-factor peptides have the structures H2N-GluAlaGluAla-alpha factor and H2N-AspAlaGluAla-alpha factor. The ste13 mutants lack a membrane-bound heat-stable dipeptidyl aminopeptidase (DPAPase A) that specifically cleaves on the carboxyl side of repeating -X-Ala- sequences. Absence of DPAPase A and the other phenotypes of a ste13 lesion cosegregate in genetic crosses. The cloned STE13 gene on a plasmid causes yeast cells to overproduce DPAPase A severalfold. A different cloned DNA segment, which weakly suppresses the ste13 defects, causes overproduction of a heat-labile activity (DPAPase B) by about tenfold. Other experiments indicate that DPAPase A action may be rate-limiting for alpha-factor maturation in normal alpha cells.     

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.     

Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist.

Mutations in the Saccharomyces cerevisiae alpha-factor receptor that lead to improved response to Saccharomyces kluyveri alpha-factor were identified and sequenced. Mutants were isolated from cells bearing randomly mutagenized receptor gene (STE2) plasmids by an in vivo screen. Five mutations lead to substitutions in hydrophobic segments in the core of the receptor (M54I, S145L, S145L-S219L, A229V, L255S-S288P). Remarkably, strains expressing these mutant receptors exhibited positive pheromone responses to desTrp1,Ala3-alpha-factor, an analog that normally blocks these responses. The M54I mutation appeared to affect only ligand specificity. The other mutations conferred additional effects on signaling or recovery. Two mutants were more sensitive to alpha-factor than wild type (S145L, A229V). One mutant was more sensitive to alpha-factor-induced cell cycle arrest initially, but then recovered more efficiently (S145L-S219L). One mutant (L255S-S288P) conferred positive pheromone responses to alpha-factor as assayed by FUS1-lacZ reporter induction, but did not display growth arrest. The hydrophobic receptor core thus appears to control activation by some ligands and to play roles in aspects of signal transduction and recovery.     

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.     

Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-alpha-factor

S. cerevisiae kex2 mutants are defective for the production of two biologically active secreted peptides: killer toxin and the mating pheromone, alpha-factor. Both molecules are excised from larger precursor polypeptides. In normal cells, the alpha-factor precursor is core-glycosylated and proteolytically processed intracellularly. In kex2 mutants, however, prepro-alpha-factor is not proteolytically cleaved and is secreted in a highly glycosylated form. All kex2 mutants examined (three independent alleles) lack a Zn++-sensitive membrane-associated endopeptidase with specificity for cleaving on the carboxyl side of a pair of basic residues. Absence of this activity cosegregates with the other phenotypes of a kex2 lesion in genetic crosses. The normal KEX2 gene was isolated by complementation of three of the phenotypes conferred by the kex2-1 mutation. The cloned DNA, either on a multicopy plasmid or integrated into the genome, restores both enzymatic activity in vitro and the normal pattern of proteolytic processing and glycosylation of prepro-alpha-factor in vivo. Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase.     

Mammalian lens dipeptidyl aminopeptidase III

An intracellular exopeptidase identified as dipeptidyl aminopeptidase III (DAP III) was found to be abundant in the bovine lens. The enzyme contained in aqueous extracts exhibited a marked preference, compared to other dipeptidyl-β-naphthylamides, for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 9.0 and 37°. The K_m for this substrate was estimated to be 2.83 × 10^?5M. Lens DAP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, and puromycin. Lens aminopeptidase activities measured at pH 7.5 on the β-naphthylamides of leucine, alanine, and arginine, included for comparison, suggested that not only is leucine aminopeptidase abundant, but also other aminopeptidases that appear to include alanine aminopeptidase and aminopeptidase B.     

Functional characterization of an alpha-factor-like Sordaria macrospora peptide pheromone and analysis of its interaction with its cognate receptor in Saccharomyces cerevisiae

The homothallic filamentous ascomycete Sordaria macrospora possesses genes which are thought to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The pheromone precursor genes are termed ppg1 and ppg2. The putative products derived from the gene sequence show structural similarity to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae. Likewise, sequence similarity has been found between the putative products of the pheromone receptor genes pre2 and pre1 and the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. To investigate whether the alpha-factor-like pheromone-receptor pair of S. macrospora is functional, a heterologous yeast assay was used. Our results show that the S. macrospora alpha-factor-like pheromone precursor PPG1 is processed into an active pheromone by yeast MATalpha cells. The S. macrospora PRE2 protein was demonstrated to be a peptide pheromone receptor. In yeast MATa cells lacking the endogenous Ste2p receptor, the S. macrospora PRE2 receptor facilitated all aspects of the pheromone response. Using a synthetic peptide, we can now predict the sequence of one active form of the S. macrospora peptide pheromone. We proved that S. macrospora wild-type strains secrete an active pheromone into the culture medium and that disruption of the ppg1 gene in S. macrospora prevents pheromone production. However, loss of the ppg1 gene does not affect vegetative growth or fertility. Finally, we established the yeast assay as an easy and useful system for analyzing pheromone production in developmental mutants of S. macrospora.     

Vps1p, a member of the dynamin GTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae.

The KEX2-encoded endoprotease of Saccharomyces cerevisiae resides in the Golgi complex where it participates in the maturation of alpha-factor mating pheromone precursor. Clathrin heavy chain gene disruptions cause mislocalization of Kex2p to the cell surface and reduce maturation of the alpha-factor precursor. Based on these findings, a genetic screen has been devised to isolate mutations that affect retention of Kex2p in the Golgi complex. Two alleles of a single genetic locus, lam1 (lowered alpha-factor maturation), have been isolated, which result in inefficient maturation of alpha-factor precursor. In lam1 cells, Kex2p is not mislocalized to the cell surface but is abnormally unstable. Normal stability is restored by the pep4 mutation which reduces the activity of vacuolar proteases. In contrast, the pheromone maturation defect is not corrected by pep4. Organelle fractionation by sucrose density gradient centrifugation shows that Kex2p is not retained in the Golgi complex of lam1 cells. Vacuolar protein precursors are secreted by lam1 mutants, revealing another sorting defect in the Golgi complex. Genetic complementation reveals that lam1 is allelic to the VPS1 gene, which encodes a dynamin-related GTPase. These results indicate that Vps1p is necessary for membrane protein retention in a late Golgi compartment.     

Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae

The role of clathrin in retention of Golgi membrane proteins has been investigated. Prior work showed that a precursor form of the peptide mating pheromone alpha-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1). This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of alpha-factor precursor, is mislocalized to the cell surface of mutant cells. We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains. Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not. In mutant cells carrying a temperature-sensitive allele of CHC1 (chc1-ts), alpha-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonpermissive temperature. In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature. Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chc1-ts cells until 2 h after the temperature shift. A temperature-sensitive mutation which blocks a late stage of the secretory pathway, sec1, prevents the appearance of mislocalized Kex2p at the cell surface of chc1-ts cells. We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.     

An unusual mutation results in the replacement of diaminopimelate with lanthionine in the peptidoglycan of a mutant strain of Mycobacterium smegmatis

Mycobacterial peptidoglycan contains L-alanyl-D-iso-glutaminyl-meso-diaminopimelyl-D-alanyl-D-alanine peptides, with the exception of the peptidoglycan of Mycobacterium leprae, in which glycine replaces the L-alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the meso-diaminopimelate (DAP) moiety of one peptide and the penultimate D-alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of Mycobacterium smegmatis that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to beta-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants, M. smegmatis strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the cbs gene, encoding cystathionine beta-synthase, an enzyme that is a part of the cysteine biosynthetic pathway.     

Saccharomyces cerevisiae mutants unresponsive to alpha-factor pheromone: alpha-factor binding and extragenic suppression.

Mutations in six genes that eliminate responsiveness of Saccharomyces cerevisiae a cells to alpha-factor were examined by assaying the binding of radioactively labeled alpha-factor to determine whether their lack of responsiveness was due to the absence of alpha-factor receptors. The ste2 mutants, known to be defective in the structural gene for the receptor, were found to lack receptors when grown at the restrictive temperature; these mutations probably affect the assembly of active receptors. Mutations in STE12 known to block STE2 mRNA accumulation also resulted in an absence of receptors. Mutations in STE4, 5, 7, and 11 partially reduced the number of binding sites, but this reduction was not sufficient to explain the loss of responsiveness; the products of these genes appear to affect postreceptor steps of the response pathway. As a second method of distinguishing the roles of the various STE genes, we examined the sterile mutants for suppression. Mating of the ste2-3 mutant was apparently limited by its sensitivity to alpha-factor, as its sterility was suppressed by mutation sst2-1, which leads to enhanced alpha-factor sensitivity. Sterility resulting from each of four ste4 mutations was suppressed partially by mutation sst2-1 or by mutation bar1-1 when one of three other mutations (ros1-1, ros2-1, or ros3-1) was also present. Sterility of the ste5-3 mutant was suppressed by mutation ros1-1 but not by sst2-1. The ste7, 11, and 12 mutations were not suppressed by ros1 or sst2. Our working model is that STE genes control the response to alpha-factor at two distinct steps. Defects at one step (requiring the STE2 gene are suppressed (directly or indirectly) by mutation sst2-1, whereas defects at the other step (requiring the STE5 gene) are suppressed by the ros1-1 mutation. The ste4 mutants are defective for both steps. Mutation ros1-1 was found to be allelic to cdc39-1. Map positions for genes STE2, STE12, ROS3, and FUR1 were determined.     

Ca2+-calmodulin promotes survival of pheromone-induced growth arrest by activation of calcineurin and Ca2+-calmodulin-dependent protein kinase.

The cmd1-6 allele contains three mutations that block Ca2+ binding to calmodulin from Saccharomyces cerevisiae. We find that strains containing cmd1-6 lose viability during cell cycle arrest induced by the mating pheromone alpha-factor. The 50% lethal dose (LD50) of alpha-factor for the calmodulin mutant is almost fivefold below the LD50 for a wild-type strain. The calmodulin mutants are not more sensitive to alpha-factor, as measured by activation of a pheromone-responsive reporter gene. Two observations indicate that activation of the Ca2+-calmodulin-dependent protein phosphatase calcineurin contributes to survival of pheromone-induced arrest. First, deletion of the gene encoding the calcineurin regulatory B subunit, CNB1, from a wild-type strain decreases the LD50 of alpha-factor but has no further effect on a cmd1-6 strain. Second, a dominant constitutive calcineurin mutant partially restores the ability of the cmd1-6 strain to survive exposure to alpha-factor. Activation of the Ca2+-calmodulin-dependent protein kinase (CaMK) also contributes to survival, thus revealing a new function for this enzyme. Deletion of the CMK1 and CMK2 genes, which encode CaMK, decreases the LD50 of pheromone compared with that for a wild-type strain but again has no effect in a cmd1-6 strain. Furthermore, the LD50 of alpha-factor for a mutant in which the calcineurin and CaMK genes have been deleted is the same as that for the calmodulin mutant. Finally, the CaMK and calcineurin pathways appear to be independent since the ability of constitutive calcineurin to rescue a cmd1-6 strain is not blocked by deletion of the CaMK genes.     

The gene encoding dipeptidyl aminopeptidase BI from Pseudomonas sp. WO24: cloning,sequencing and expression in Escherichia coli

We have isolated the dipeptidyl aminopeptidase BI (DAP BI) gene from the plasmid library of Pseudomonas sp. WO24 chromosomal DNA by the enzymatic plate asaay using a chromogenic substrate. The DAP BI gene, designated dap b1, was further subcloned and sequenced. Sequence analysis of an approx. 3-kb fragment revealed an open reading frame of 2169 nucleotides, which was assigned to the dap b1 gene by N-terminal and internal amino acid sequences. The predicted amino acid sequence of DAP BI containing a serine protease Gly–X–Ser–X–Gly consensus motif displays extensive homologies to the several proteases belonging to the prolyl oligopeptidase family, a novel serine protease family possessing the catalytic triad with a specific array of Ser, Asp and His in this order, which is the hallmark of the member of this family including DAP IV. The dap b1 gene was expressed in Escherichia coli and the expressed enzyme was purified about 230-fold with 2.6% recovery from the cell-free extracts. The enzymatic properties such as molecular mass, substrate specificity and effect of inhibitor were similar to the native enzyme from Pseudomonas sp. WO24.     

Cytochemical localization and biochemical characterization of dipeptidyl aminopeptidase II in macrophages and mast cells

Dipeptidyl aminopeptidase II (DAP II) was demonstrated cytochemically at light and electron microscope levels in rat macrophages and mast cells using Lys-Ala-4-methoxy-2-naphthylamide as a specific substrate. The enzyme which was found to be lysosomal in both cell types, was analyzed biochemically in extracts by measuring fluorometrically the liberated naphthylamine, and was visualized in sections microscopically using azo-coupling methods. DAP II was further characterized by isoelectric focusing techniques. Macrophage DAP II was found to be typical of that found in other rat tissues in terms of its structural latency, substrate specificity, inhibitor sensitivities, and pH activator requirements. Addition DAP II isozymes, not previously recognized, were observed.