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PubMed1983

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.

Amino Acids↗

PubMed1990

Calmodulin-binding proteins of Saccharomyces cerevisiae.

The subcellular distribution of calmodulin-binding proteins in the soluble, plasma membrane, and nuclear fractions of Saccharomyces cerevisiae was analyzed with a gel binding assay using 125I-labeled calmodulin. Over 20 binding proteins were detected. The calmodulin-binding protein profiles were markedly different among the fractions. Calmodulin-binding proteins were most abundant in the nuclear fraction, followed by the membrane fraction and the soluble fraction in decreasing order. The amounts of certain calmodulin-binding proteins increased after treatment with alpha-mating factor.

Calmodulin↗

PubMed1993

Expression of A mating type genes of Coprinus cinereus in a heterologous basidiomycete host.

The A mating factor of Coprinus cinereus determines compatibility in mating by regulating part of a developmental sequence that leads to dikaryon formation. The A genes that trigger development encode two different classes of homeodomain proteins, and for a successful mating, a protein of one class, HD1, must interact with a protein of the other class, HD2. In this report we show that C. cinereus A genes that encode HD2 proteins, a2-1 and b2-1, can elicit A-regulated development in the heterologous host C. bilanatus. Transformation rates were very low, suggesting that the genes were poorly transcribed. The fact that the HD2 genes are functionally expressed implies successful heteromultimeric association of putative DNA-binding proteins coded by the two Coprinus species. This interaction was sufficient to satisfy the need for different A factors in the formation of a fertile C. bilanatus dikaryon, but fertile dikaryons were more readily produced in matings with the a2-1 gene transformants. The C. cinereus A genes, b1-1 and d1-1, which encode HD1 proteins, were either not expressed or their proteins were non-functional in C. bilanatus. These experiments raise some interesting questions regarding HD1-HD2 protein interactions.

Cloning, Molecular↗

PubMed2001

The ste3 pheromone receptor gene of Pneumocystis carinii is surrounded by a cluster of signal transduction genes.

Although the clinical aspects of Pneumocystis carinii pneumonia are well characterized, the basic biology of the causative organism is poorly understood. Most proposed life cycles of P. carinii include both asexual and sexual replicative cycles. The two most prominent morphological forms are a trophic form, thought to undergo asexual replication by binary fission, and a cystic form or ascus containing intracystic bodies or ascospores, the products of sexual replication. To facilitate the Pneumocystis genome project, a P. carinii f. sp. carinii genomic cosmid library and an additional lambda cDNA library were generated. A partial expressed sequence tag database, created as part of the genome project, revealed the transcription of meiosis-specific genes and other genes related to sexual reproduction. The ortholog of Ste3, an a-factor pheromone receptor, was cloned and genes surrounding the ste3 locus were examined. Clustered around the ste3 gene are genes encoding elements functional in the pheromone response signal transduction cascade of model fungal organisms. These include the Ste20 protein kinase, the Ste12 homoeodomain transcriptional regulator, a potential pheromone mating factor, and other DNA-binding proteins. The genomic organization of the ste3 locus bears significant similarity to that of the mating locus recently described in Cryptococcus neoformans. The P. carinii genome contains much of the genetic machinery necessary for pheromone responsiveness, and these data support the existence of a sexual replication cycle.

Amino Acid Sequence↗

PubMed1994

Consequences of altered isoprenylation targets on a-factor export and bioactivity.

Cysteine-containing amino acid sequences (CAAX, CC, and CXC; C is cysteine, A is any aliphatic amino acid, and X is any amino acid) are targets for the attachment of C15 (farnesyl) and C20 (geranylgeranyl) isoprenoids to peptides and proteins by specific prenyltransferases. Although much work has centered on the enzymatic mechanisms of these enzymes, the biological consequences of the differential isoprenylation they catalyze remain to be elucidated. Farnesylation of the a-factor mating pheromone of Saccharomyces cerevisiae is a known prerequisite for its biological activity and its secretion through a pathway utilizing the yeast STE6 protein, a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein. We generated specific mutations in the a-factor gene to encode isoprenylation targets for geranylgeranylation [Cys-Val-Ile-Leu (CVIL) and Ser-Val-Cys-Cys (SVCC)] in place of the natural farnesylation motif [Cys-Val-Ile-Ala (CVIA)]. The a-factors containing these modified prenylation sites were successfully exported by a STE6-dependent mechanism. Furthermore, these peptides, as well as synthetic geranylgeranyl a-factor, retained bioactivity. Chromatographic comparisons of synthetic and biosynthetic pheromones suggest that, in vivo, a peptide substrate containing the geranylgeranylation target CVIL can be both farnesylated and geranylgeranylated. These results clearly demonstrate that in vivo (i) different prenyltransferases may recognize the same substrate; (ii) both farnesylated and geranylgeranylated a-factor peptides are substrates for export via STE6, a MDR-like protein; and (iii) farnesylated and geranylgeranylated pheromones are both biologically active.

ATP-Binding Cassette Transporters↗

PubMed1992

Production of a soluble and secreted antigenic fragment of HBsAg in yeast.

We have produced a fragment of hepatitis B surface antigen (HBsAg) corresponding to amino acids 1-60 as a fusion protein with the alpha mating factor of yeast. The product is secreted from yeast as a soluble monomer that expresses HBsAg antigenicity. Unlike other heterologous fusion proteins, it is not processed by the Lys-Arg endoprotease, possibly due to a proline in the linker between the two coding sequences. The resulting soluble fragment will enable us to map the immunodominant sites of HBsAg recognized by T cells and to identify additional factors contributing to vaccine potency.

Amino Acid Sequence↗

PubMed1990

Cell cycle arrest caused by CLN gene deficiency in Saccharomyces cerevisiae resembles START-I arrest and is independent of the mating-pheromone signalling pathway.

Null mutations in three genes encoding cyclin-like proteins (CLN1, CLN2, and CLN3) in Saccharomyces cerevisiae cause cell cycle arrest in G1 (cln arrest). In cln1 cln2 cln3 strains bearing plasmids containing the CLN3 (also called WHI1 or DAF1) coding sequence under the transcriptional control of a galactose-regulated promoter, shift from galactose to glucose medium (shutting off synthesis of CLN3 mRNA) allowed completion of cell cycles in progress but caused arrest in the ensuing unbudded G1 phase. Cell growth was not inhibited in arrested cells. Cell division occurred in glucose medium even if cells were arrested in S phase during the initial 2 h of glucose treatment, suggesting that CLN function may not be required in the cell cycle after S phase. However, when the coding sequence of the hyperactive C-terminal truncation allele CLN3-2 (formerly DAF1-1) was placed under GAL control, cells went through multiple cycles before arresting after a shift from galactose to glucose. These results suggest that the C terminus of the wild-type protein confers functional instability. cln-arrested cells are mating competent. However, cln arrest is distinct from constitutive activation of the mating-factor signalling pathway because cln-arrested cells were dependent on the addition of pheromone both for mating and for induction of an alpha-factor-induced transcript, FUS1, and because MATa/MAT alpha (pheromone-nonresponsive) strains were capable of cln arrest in G1 (although a residual capacity for cell division before arrest was observed in MATa/MAT alpha strains). These results are consistent with a specific CLN requirement for START transit.

Alleles↗

PubMed1989

Rapid intracellular alkalinization of Saccharomyces cerevisiae MATa cells in response to alpha-factor requires the CDC25 gene product.

The alpha-factor mating pheromone induces a transient intracellular alkalinization of MATa cells within minutes after exposure to the pheromone, and is the earliest biochemical event that can be identified subsequent to the exposure. Dissipation of the pheromone induced pH gradient, using 2,4-dinitrophenol or sodium orthovanadate, does not inhibit the biological response of the yeast to the pheromone such as mating and 'schmoo' formation. These findings suggest that the pheromone mediated pH change per se is not a part of the transmembrane signalling but rather the consequence of a biochemical reaction triggered by the alpha-pheromone interaction with its receptor and may have a permissive effect on the pheromonal response. The cdc25ts mutation causes MATa cells to become nonresponsive to alpha-factor subsequent to a shift to the restrictive temperature, suggesting that the CDC25 gene product participates in the pheromone response pathway.

Cell Cycle Proteins↗

PubMed2001

The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor.

Saccharomyces cerevisiae Ste24p is a multispanning membrane protein implicated in the CAAX proteolysis step that occurs during biogenesis of the prenylated a-factor mating pheromone. Whether Ste24p acts directly as a CAAX protease or indirectly to activate a downstream protease has not yet been established. In this study, we demonstrate that purified, detergent-solubilized Ste24p directly mediates CAAX proteolysis in a zinc-dependent manner. We also show that Ste24p mediates a separate proteolytic step, the first NH(2)-terminal cleavage in a-factor maturation. These results establish that Ste24p functions both as a bona fide COOH-terminal CAAX protease and as an a-factor NH(2)-terminal protease. Importantly, this study is the first to directly demonstrate that a eukaryotic multispanning membrane protein can possess intrinsic proteolytic activity.

Amino Acid Sequence↗

PubMed1997

Position one analogs of the Saccharomyces cerevisiae tridecapeptide pheromone.

Analogs of the Saccharomyces cerevisiae alpha-mating factor [WHWLQLKPGQPMY], in which a variety of residues replaced Trp1 were synthesized and assayed for biological activity and receptor affinity. Analogs containing Gly or Leu or many different aromatic residues in position 1 of the peptide exhibited bioactivity in a growth arrest assay slightly greater than, or equal to, that of the parent pheromone, whereas the Glu1 and Lys1 analogs exhibited significantly lower bioactivity. Analogs with an aromatic replacement at position 1 had 3- to 6-fold lower receptor affinity than the parent peptide, whereas analogs with a hydrophilic residue at the N-terminus exhibited large reductions in receptor affinity with the peptide with Glu in position 1 showing a 120-fold reduction. N alpha-Acetylation had little effect on bioactivity but lowered receptor affinity by 20- to 40-fold. Amidation of the carboxyl terminus resulted in a 10-fold decrease in activity and a 160-fold decrease in receptor affinity. These results indicate that the alpha-factor receptor has a large hydrophobic binding pocket, possibly containing a negatively charged side-chain, which interacts with the N-terminus of alpha-factor. The lack of correlation between activity and binding and several analogs suggests that small residues near the N-terminus of alpha-factor may be very efficient in triggering isomerization of the receptor to its activated state in the first step of the signal transduction pathway.

Gene Expression↗

PubMed1992

Endoproteolytic processing of a farnesylated peptide in vitro.

Numerous eukaryotic proteins containing a carboxyl-terminal CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) require a three-step posttranslational processing for localization and function. The a mating factor of Saccharomyces cerevisiae is one such protein, requiring cysteine farnesylation, proteolysis of the terminal three amino acids, and carboxyl methylation for biological activity. We have used farnesylated a-factor peptides to examine the proteolytic step in the maturation of CAAX-containing proteins. Three distinct carboxyl-terminal protease activities were found in yeast cell extracts that could remove the terminal three residues of a-factor. Two of the proteolytic activities were in cytosolic fractions. One of these activities was a PEP4-dependent carboxypeptidase that was sensitive to phenylmethylsulfonyl fluoride. The other cytosolic activity was PEP4-independent, sensitive to 1,10-phenanthroline, and effectively inhibited by an unfarnesylated a-factor peptide. In contrast, a protease activity in membrane fractions was unaffected by phenylmethylsulfonyl fluoride, 1,10-phenanthroline, or unfarnesylated a-factor peptide. Incubation of membrane preparations from either yeast or rat liver with a radiolabeled farnesylated a-factor peptide released the terminal three amino acids intact as a tripeptide, indicating that this reaction occurred by an endoproteolytic mechanism and that the enzyme most likely possesses a broad substrate specificity. The yeast endoprotease was not significantly affected by a panel of protease inhibitors, suggesting that the enzyme is novel. Zinc ion was shown to inhibit the endoprotease (Ki less than 100 microM). The specific activities of the a-factor carboxyl-terminal membrane endoprotease and methyltransferase clearly indicated that the proteolytic reaction was not rate-limiting in these processing reactions in vitro.

Amino Acid Sequence↗

PubMed1995

Systematic analysis of the Saccharomyces cerevisiae alpha-factor containing lactam constraints of different ring size.

Eight cyclic analogs and corresponding linear homologs of the alpha-factor mating pheromone (WHWLQLKPGQPMY) of Saccharomyces cerevisiae were synthesized using solid-phase procedures on a phenylacetamidomethyl support. On-resin lactamization of the side chains of residues 7 and 10 to form rings containing from 14 to 18 atoms was effected by the BOP reagent. All peptides were highly homogeneous and gave expected molecular ions by FAB mass spectrometry. The constrained analogs had biological activities varying from 10% to less than 0.1% of that of [Nle12]-alpha-factor. In all cases, cyclic analogs with Glu in position 10 were more active than the homolog with Asp at this position. This trend was also found with the corresponding linear pheromones, suggesting that a gamma-carbonyl in position 10 is an important determinant of pheromone potency. The cyclic peptides had from 50- to 20000-fold lower affinities for the alpha-factor receptor than for [Nle12]-alpha-factor, as judged using a competition binding assay. Circular dichroism studies indicate that the cyclic lactam-containing region of cyclo7.10[Orn7, Glu10,Nle12]-alpha-factor retains a beta-turn-like structure similar to that found in the corresponding model tetrapeptide. The results show that covalently constrained analogs of the linear pheromone can maintain biological activity, despite binding poorly to the receptor, and indicate that a beta-turn-like structure in the center of the pheromone allows signal transduction.

Binding, Competitive↗

PubMed1991

SRH1 protein, the yeast homologue of the 54 kDa subunit of signal recognition particle, is involved in ER translocation of secretory proteins.

The function of the SRH1 product, the yeast homologue of the 54 kDa subunit of the mammalian signal recognition particle, has been analyzed using a galactose dependent mutant of the gene. SRH1 has been placed under control of the GAL1 promoter and introduced into a haploid cell that had its chromosomal SRH1 copy disrupted. This mutant grows normally on galactose medium but slows down the growth about 10 h after transfer to glucose medium. At the same time, precursor forms of secretory proteins, alpha-mating factor and invertase, accumulate in the cells. This result indicates that the SRH1 product is involved in translocation of precursors of secretory proteins across the endoplasmic reticulum membrane in yeast cells.

Base Sequence↗

PubMed1988

STE2 protein of Saccharomyces kluyveri is a member of the rhodopsin/beta-adrenergic receptor family and is responsible for recognition of the peptide ligand alpha factor.

We have cloned the gene for the alpha-factor receptor of the yeast Saccharomyces kluyveri by using the Saccharomyces cerevisiae receptor gene (c-STE2) as a probe. The nucleotide sequence of the S. kluyveri gene (k-STE2) shows that its predicted polypeptide contains seven hydrophobic segments capable of spanning a lipid bilayer and thus that, like c-STE2, it appears to be a member of the rhodopsin/beta-adrenergic receptor family. The k-STE2 polypeptide is 50% identical to that coded by c-STE2, with high conservation (greater than 67%) in the putative membrane-spanning domains. The carboxyl-terminal amino acid sequences are not similar, but both are very hydrophilic and rich in serine and threonine residues. The k-STE2 gene is functional in S. cerevisiae: it reverses the mating defect of an S. cerevisiae mutant defective in its STE2 gene. S. cerevisiae strains expressing k-STE2 rather than c-STE2 exhibit the mating-factor selectivity characteristic of S. kluyveri: better response to S. kluyveri alpha factor than to S. cerevisiae alpha factor. (S. cerevisiae normally responds much better to its own alpha-factor peptide than to the related alpha-factor peptide of S. kluyveri.) This observation demonstrates that the STE2 gene is responsible for ligand selectivity and provides additional evidence that the STE2 protein is the receptor for alpha factor.

Amino Acid Sequence↗

PubMed1990

Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae.

Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells. Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity. This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures. We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell. To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced. Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme. Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.

Amino Acid Sequence↗

PubMed1990

Prenyl proteins in eukaryotic cells: a new type of membrane anchor.

Recent studies have indicated that eukaryotic cells contain proteins that are post-translationally modified by long-chain, thioether-linked prenyl groups. These proteins include yeast mating factors, ras proteins and nuclear lamins. The modification occurs on a cysteine residue near the C terminus and appears to initiate a set of additional protein modification reactions that promote attachment of the proteins to specific membranes.

Animals↗

PubMed1994

NMR investigation of cyclo7,10[C7,X9,C10,Nle12] analogues of the alpha-factor from Saccharomyces cerevisiae.

The cyclo7,10[Cys7,Cys10,Nle12], cyclo7,10[Cys7,D-Ala9,Cys10,Nle12], and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] analogues of the alpha-factor mating pheromone (WHWLQLKPGQPMY) of the yeast Saccharomyces cerevisiae were studied in DMSO/water (80:20) and aqueous solution by nmr spectroscopy. In addition, the cyclo7,10[Cys7,D-Val9,Cys10,Nle12]alpha-fa ctor was examined in DMSO/water. Nuclear Overhauser effect (NOE) and NH d delta/dT data indicate that the cyclo7,10[Cys7,D-Val9,Cys10,Nle12]alpha-fa ctor adopts a type II beta-turn in DMSO/water and that the cyclo7,10[Cys7,D-Ala9,Cys10,Nle12]- and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12]alpha-fa ctor analogues adopt type II and type I/III beta-turns, respectively, in both DMSO/water and aqueous solutions. In aqueous solution, residues 8 and 9 of the cyclo7,10[Cys7,Nle12] alpha-factor appear to adopt at least two distinct conformations, one of these being identified as a type I/III beta-turn. In contrast, the cyclo7,10[Cys7,Cys10,Nle12] alpha-factor appears to adopt predominately a type II beta-turn in DMSO/water. Quantitative NOE measurements of the cyclo7,10[Cys7,Cys10,Nle12]-, cyclo7,10[Cys7,D-Val9,Cys10,Nle12]-, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factors in DMSO/water were used to derive three-dimensional structures of the cyclo7,10[Cys7,Pro8,X9,Cys10] portion of these analogues.

Amino Acid Sequence↗

PubMed2003

Human TSG101 does not replace Saccharomyces cerevisiae VPS23 role in the quality control of plasma membrane proteins.

The Saccharomyces cerevisiae VPS23 (STP22) gene is implicated in the control of vesicle movement and quality of plasma membrane proteins. VPS23 mutants have defects either in removing defective membrane proteins such as alpha-mating factor receptor and arginine permease. The human ortholog TSG101 and its variants, isolated from tumor cells, do not substitute VPS23 in its ability to rescue the phenotype of defective plasma membrane proteins.

Arginine↗