The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae

The STE2 gene of Saccharomyces cerevisiae encodes a 431-residue protein containing seven hydrophobic segments that is thought to be an essential component of the cell-surface receptor for alpha-factor in MATa haploids. Methods were devised to prepare membrane fractions from MATa cells that retained high levels of alpha-factor binding activity, consistent with the view that the alpha-factor receptor resides in the plasma membrane. To demonstrate that the membrane constituent responsible for alpha-factor binding was the STE2 polypeptide, specific antibodies were generated and used to identify STE2-related polypeptides by radiolabeling, immunoprecipitation, and polyacrylamide gel electrophoresis. Under conditions of complete solubilization, the major form of the STE2 gene product detected was a glycoprotein with an apparent molecular weight of 49,000. Affinity labeling of yeast membrane preparations by chemical cross-linking to 35S-alpha-factor indicated that a molecule of 49,000 molecular weight was the major alpha-factor-binding species. This alpha-factor-binding species was shown to be the product of the STE2 gene in three ways. First, MATa haploids carrying the STE2 gene on a multicopy plasmid overproduced alpha-factor binding activity about 15-fold. Second, MATa cells completely lacking a STE2 gene showed only nonspecific binding of alpha-factor (equivalent to the level displayed by MAT alpha haploids) and possessed no species that could be cross-linked to 35S-alpha-factor. Third, MATa cells expressing a truncated but functional STE2 gene (in which the COOH-terminal 135-hydrophilic residues were deleted) produced a protein detected by cross-linking to 35S-alpha-factor of apparent molecular weight 33,000, close to the size expected for the predicted abbreviated STE2 polypeptide. These findings demonstrate unequivocally that the STE2 gene product is the membrane component responsible for the ligand recognition function of the yeast alpha-factor receptor.     

Glycosylation and structure of the yeast MF alpha 1 alpha-factor precursor is important for efficient transport through the secretory pathway

The MF alpha 1 gene encodes a precursor, prepro-alpha-factor, that undergoes several proteolytic processing steps within the classical secretory pathway to produce the mature peptide pheromone, alpha-factor. To investigate the role of structural features of the MF alpha 1 precursor in alpha-factor production, we analyzed the effect of mf alpha 1 mutations that alter precursor structure in a number of ways. These mutations resulted in decreased alpha-factor secretion and intracellular accumulation of pro-alpha-factor. With the exception of the mutant lacking all three N glycosylation sites, the pro-alpha-factor forms that accumulated were core glycosylated but had not yet undergone the addition of outer chain carbohydrate. The delay, therefore, occurred at a step prior to the first proteolytic processing step involved in maturation of the precursor and was probably due to inefficient endoplasmic reticulum-to-Golgi transport. Elimination of all three N-glycosylation sites caused a delay in disappearance of intracellular precursor, and alpha-factor secretion was also slowed. These data indicate that N glycosylation is important but not essential for transport of the precursor through the secretory pathway. The decreased alpha-factor secretion and increased precursor accumulation seen with many different structural changes of pro-alpha-factor indicate that the secretory pathway is extremely sensitive to changes in precursor structure. This sensitivity could cause inefficient secretion of heterologous proteins and hybrids between MF alpha 1 and heterologous proteins in yeast cells.     

Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor

We have cloned and sequenced a gene (MF alpha) coding for alpha-factor, a tridecapeptide mating factor secreted by yeast alpha cells. A plasmid carrying the MF alpha gene was identified by screening for production of alpha-factor by mat alpha 2 mutants, which fail to secrete alpha-factor because of simultaneous synthesis and degradation of the factor. The cloned segment codes for four mature alpha-factor within a putative precursor of 165 amino acids. The putative precursor begins as a signal sequence for secretion. The next segment, of approximately 60 amino acids, contains three potential glycosylation sites. The carboxy-terminal half of the precursor contains four tandem copies of mature alpha-factor, each preceded by spacer peptides of six or eight amino acids (variations of Lys-Arg-Glu-Ala-Asp-Ala-Glu-Ala), which are hypothesized to contain proteolytic processing signals.     

Hormone processing and membrane-bound proteinases in yeast.

A search for maturating peptidases of the precursor protein of the mating hormone (pheromone) alpha-factor of Saccharomyces cerevisiae was performed using short model peptides representing those sequences of the precursor protein, where cleavage is thought to occur in vivo. This search was done in a mutant lacking several of the unspecific vacuolar peptidases. The chromogenic peptide Cbz-Tyr-Lys-Arg-4-nitroanilide led to the detection of a membrane-bound enzyme called proteinase yscF. Cleavage of the synthetic peptide derivative occurs after the basic amino acid pair, a proposed signal for hormone processing. Optimum pH for the reaction is 7.2. The enzyme does not cleave after single basic amino acid residues indicating that it is distinct from trypsin-like proteinases. Proteolytic activity is enhanced by Triton X-100. The enzyme is strongly inhibited by EGTA, EDTA and mercurials but insensitive to phenylmethylsulfonyl fluoride. The enzyme activity is strongly dependent on Ca2+ ions. In a mutant (kex2), which accumulates an over-glycosylated alpha-factor precursor, no proteinase yscF activity can be found. Membrane-bound peptidase activity possibly involved in removal of the arginyl and lysyl residues remaining at the carboxy terminus of the alpha-factor pheromone peptide after the initial cut of the precursor molecule could be identified by using the model peptides Cbz-Tyr-Lys-Arg and Cbz-Tyr-Lys.     

Subcellular location of enzymes involved in the N-glycosylation and processing of asparagine-linked oligosaccharides in Saccharomyces cerevisiae

A particulate translation system isolated from the yeast Saccharomyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-alpha-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-alpha-factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-alpha-F0) differing in molecular mass from a cytosolically located, protease-K-sensitive alpha-factor polypeptide (pp-alpha-Fcyt) by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated alpha-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product pp-alpha-Fcyt but larger than the membrane-specific pp-alpha-F0. Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-alpha-factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase II and a trimming mannosidase, which process the prepro-alpha-factor glycans down to the (Man)8(GlcNAc)2 stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes.     

Porcine growth hormone: molecular cloning of cDNA and expression in bacterial and mammalian cells

Porcine growth hormone (PGH) precursor cDNAs were cloned from a pituitary cDNA library constructed in lambda gt11 by immunoscreening. One of the three clones characterized contained an entire nucleotide sequence for the 216-amino-acid precursor molecule. The deduced amino-acid sequence of PGH confirmed the sequence previously reported for that of the genomic DNA of PGH except for one base difference in the coding sequence. Expression of the full-length PGH cDNA was achieved in bacteria and mammalian cells. The mammalian cell line, COS-1, produced the GH molecule which processed the signal peptide and had the same molecular weight as standard PGH, in contrast to the higher molecular weight of the bacterial product. Radioimmunoassay of the recombinant PGH produced in COS-1 cells also revealed an inhibition curve similar to that of the standard PGH.     

Secretion of somatostatin by Saccharomyces cerevisiae. Correct proteolytic processing of pro-alpha-factor-somatostatin hybrids requires the products of the KEX2 and STE13 genes

Somatostatin is a 14-amino-acid peptide hormone that is proteolytically excised from its precursor, prosomatostatin, by the action of a paired-basic-specific protease. Yeast (Saccharomyces cerevisiae Mat alpha) synthesizes an analogous peptide hormone precursor, pro-alpha-factor, which is proteolytically processed by at least two separate proteases, the products of the KEX2 and STE13 genes, to generate the mature bioactive peptide. Expression in yeast of recombinant DNAs encoding hybrids between the proregion of alpha-factor and somatostatin results in proteolytic processing of the chimeric precursors and secretion of mature somatostatin. To determine if the chimeras were processed by the same enzymes that cleave endogenous pro-alpha-factor, the hybrid DNAs were introduced into kex2 and ste13 mutants, and the secreted proteins were analyzed. Expression of the pro-alpha-factor-somatostatin hybrids in kex2 mutant yeast resulted in secretion of a high molecular weight hyperglycosylated precursor. No mature somatostatin was secreted, and there was no proteolytic cleavage at the Lys-Arg processing site. Similarly, in ste13 yeast, only somatostatin molecules containing the (Glu-Ala)3 spacer peptide at the amino terminus were secreted. Our results demonstrate that in yeast processing mutants, the behavior of the chimeric precursors with respect to proteolytic processing was exactly as that of endogenous pro-alpha-factor. We conclude that the same enzymes that generate mature alpha-factor proteolytically process hybrid precursors. This suggests that structural domains of the proregion rather than the mature peptide are recognized by the processing proteases.     

Biochemical and mass spectrometric characterization of the human CB2 cannabinoid receptor expressed in Pichia pastoris--importance of correct processing of the N-terminus

This study was conducted to optimize the expression of human CB2 cannabinoid receptors in methylotrophic yeast Pichia pastoris (P. pastoris). Two major species of expressed CB2 proteins were seen on Western blot, i.e., a 42 kDa band which matches the calculated molecular weight for tagged CB2, and a 52/55 kDa doublet. Treatment of membranes with N-glycosidase F or inclusion of tunicamycin in the culture medium during induction resulted in the disappearance of the 55 kDa, but not the 52 kDa band, suggesting that the 3 kDa extra in the 55 kDa band is due to N-glycosylation, but the 10 kDa extra in the 52 kDa band is not due to N-glycosylation. Anti-FLAG M1 antibody had a much higher preference for the 42 kDa band over the 52/55 kDa doublet, and a 10 kDa fragment recognized by anti-FLAG M2 antibody was generated by CNBr digestion of the 52/55 doublet. These data strongly support the hypothesis that the 10 kDa increase in molecular weight was due to unprocessed alpha-factor sequence. This conclusion was further validated by finding several peptide sequences for alpha-factor fragments at the N-terminal of the CB2 receptor using pepsin/chymotrypsin digestion and LC/MS/MS approaches. Importantly, unprocessed alpha-factor was found to be associated with poor ligand binding. In addition, controlling the level of CB2 protein expression was found to be critical for minimizing the presence of unprocessed alpha-factor sequence. The information gained from this study should aid the proper expression of not only CB2 receptor but also other members of the GPCR family in P. pastoris.     

Production of full-length human pre-elafin, an elastase specific inhibitor, from yeast requires the absence of a functional yapsin 1 (Yps1p) endoprotease

Pre-elafin, also known as trappin-2, is an elastase-specific inhibitor that belongs to the trappin gene family. A chimeric gene encoding polyhistidine-tagged human pre-elafin fused to the yeast alpha-factor precursor was expressed in Saccharomyces cerevisiae. The chimera was engineered to keep a single copy of the mature alpha-factor peptide. This enabled the use of a simple bioassay (mating assay) to assess the relative efficiency of both the expression and the secretion of the recombinant molecule. We found that pre-elafin is processed both in vivo and in vitro by yapsin 1, the yeast aspartyl endoprotease encoded by YPS1. Cleavage by yapsin 1 occurred C-terminal to a subset of single lysine residues. Expression in a yapsin 1-deficient yeast strain was an indispensable condition to allow the efficient production of full-length human pre-elafin. The recombinant inhibitor was purified from concentrated culture medium by ammonium sulfate precipitation, affinity purification on a Ni(2+) resin, and cation exchange chromatography. Recombinant human pre-elafin was fully active and showed the same inhibitory profile toward different serine proteases to that reported for mature elafin.     

Isolation and partial characterization of a low molecular weight trypsin inhibitor from human urine

A low molecular weight glycoprotein which completely inhibited trypsin at a 1 : 1 molar ratio was isolated from human urine. It was generated from a precursor molecule which in turn derived from plasma inter-alpha-trypsin inhibitor. It had one polypeptide chain with a molecular weight of about 20 000 and a high content of half-cystine residues. Its amino-terminal amino-acid sequence was Val-Thr-Glu-Val-Thr-X-Leu-Glu-Asp-.     

Expression and secretion of biologically active human atrial natriuretic peptide in Saccharomyces cerevisiae

A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor. Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium. The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus. The secreted product was also folded correctly with respect to the single disulfide bond. However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material. The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.     

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.     

Heterologous expression of peptide hormone precursors in the yeast Saccharomyces cerevisiae. Evidence for a novel prohormone endoprotease with specificity for monobasic amino acids.

The peptide somatostatin (SRIF) exists as two different molecular species. In addition to the most common form, which is a 14-residue peptide, there is also a 14-amino acid amino-terminally extended form of the tetradecapeptide, SRIF-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which, upon cleavage, generate either SRIF-14 or -28, respectively. In mammals a single prepro-SRIF molecule undergoes tissue-specific processing to generate the mature hormone whereas in some species of fish separate genes encode two distinct but homologous precursors prepro-SRIF-I and -II that give rise to SRIF-14 and -28, respectively. To investigate the molecular basis for differential processing of the prohormones we introduce their cDNAs into yeast cells (Saccharomyces cerevisiae). The signal peptides of both precursors were poorly recognized by the yeast endoplasmic reticulum translocation apparatus, consequently only low levels of SRIF peptides were synthesized. To circumvent this problem a chimeric precursor consisting of the alpha-factor signal peptide plus 30 residues of the proregion was fused to pro-SRIF-II. This fusion protein was efficiently transported through the yeast secretory pathway and processed to SRIF-28 exclusively, which is identical to the processing of the native precursor in pancreatic islet D-cells. Most significantly, cleavage of the precursor to SRIF-28 was independent of the Kex 2 endoprotease since processing occurred efficiently in a kex 2 mutant strain. We conclude that in addition to the Kex 2 protease, yeast possess a distinct prohormone converting enzyme with specificity toward monobasic processing sites.     

Tyrosine phosphorylation of a yeast 40 kDa protein occurs in response to mating pheromone.

Tyrosine phosphorylation of proteins in the yeast Saccharomyces cerevisiae has been examined following exposure to the mating pheromone alpha-factor. When a cells are treated with alpha-factor a protein of approximately 40 kDa molecular weight is tyrosine phosphorylated. This tyrosine phosphorylation response requires an intact signal transduction pathway, is not restricted to a short interval of the cell division cycle, and requires protein synthesis for its maximal accumulation. Mating competent fus3 deletion strains fail to elaborate the phosphotyrosine response. The possibility that FUS3 encodes the 40 kDa protein is discussed.     

Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway

Events in the synthesis and processing of prepro-alpha-factor have been assessed with the aid of mutants blocked at various stages in the yeast secretory pathway. In normal cells treated with tunicamycin, a precursor accumulates which is identical in molecular weight to the primary translation product synthesized in vitro. At the restrictive temperature in a mutant blocked early in the pathway (sec53), a molecule of similar molecular weight accumulates. In mutants affecting translocation into (sec59) and passage from (sec 18) the endoplasmic reticulum, a glycosylated form of the precursor containing three N-linked core oligosaccharides accumulates; however, it appears that the signal peptide is not removed. The glycosylated precursor first experiences proteolytic processing when accumulated in a mutant (sec7) blocked at the stage of the Golgi apparatus. Substantially greater amounts of the mature pheromone are seen in mutants that accumulate secretory vesicles (sec1, sec2, sec3, sec5).     

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.     

Incomplete process of recombinant human platelet-derived growth factor produced in yeast and its effect on the biological activity.

Partially purified recombinant human Platelet-derived Growth Factor BB homodimer isolated from yeast culture media contains variable amounts of unprocessed PDGF-BB. This unprocessed PDGF-BB is found as a result of incomplete cleavage of the precursor to form the mature protein. Although the signal peptide is efficiently removed, a fraction of the PDGF secreted has an extended sequence corresponding to the truncated yeast alpha-factor leader. The data suggest that it is the amino acid chain from the truncated a-factor leader and not the sugar moiety attached to it that is responsible for the higher mitogenic activity found in this unprocessed molecule compared to highly purified PDGF-BB.     

Biological activity of the Asn-5,Arg-7 tridecapeptide encoded by MF alpha 2 of Saccharomyces cerevisiae.

The precursor predicted by the nucleotide sequence of the MF alpha 2 gene of Saccharomyces cerevisiae contains one copy of the tridecapeptide alpha-factor previously characterized (H2N-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-COOH) and one copy of a peptide that contains two conservative amino acid substitutions (H2N-Trp-His-Trp-Leu-Asn-Leu-Arg-Pro-Gly-Gln-Pro-Met-Tyr-COOH). To determine whether the novel molecule possesses biological activity, the Asn-5,Arg-7 tridecapeptide was prepared chemically by solid-phase peptide synthesis. Growth arrest and morphogenesis assays gave identical activity profiles for the Asn-5,Arg-7 peptide and the other gene product, the Gln-5,Lys-7 peptide. The activities of the two peptides were additive and indistinguishable for S. cerevisiae X2180-1A. When present in fourfold molar excess, the biologically inactive desTrp-1,Ala-3 dodecapeptide reversed activity of the Asn-5,Arg-7 and Gln-5,Lys-7 tridecapeptides. Furthermore, neither peptide caused growth arrest of a MATa ste2(Ts) mutant when assayed at the restrictive temperature. These studies suggest that both pheromones interact with the alpha-factor receptor in a similar manner.     

Cleavage of human fibroblast type I procollagen by mammalian collagenase: demonstration of amino- and carboxy-terminal extension peptides.

Human skin fibroblasts in monolayer culture synthesize and secrete precursor forms of collagen into the culture medium. The type I collagen precursor, the major precursor in the culture medium, was isolated on DEAE cellulose chromatography and subjected to mammalian collagenase cleavage. The amino terminal cleavage fragments had a higher molecular weight than α1^A and α2^A, but did not contain interchain disulfide bonds. The carboxy-terminal cleavage fragments formed high molecular weight aggregates which contained interchain disulfide bonds. These results indicate that human type I procollagen contains noncollagenous amino and carboxy-terminal extension peptides and that all of the interchain disulfide bonds are on the carboxy-terminal portion of the molecule.