Name length as a factor in mate selection.
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Behavioral, social and psychological factors affecting mating time and breeding efficiency in sheep.
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Animals↗
Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating.
The role of alpha-factor structural genes MF alpha 1 and MF alpha 2 in alpha-factor production and mating has been investigated by the construction of mf alpha 1 and mf alpha 2 mutations that totally eliminate gene function. An mf alpha 1 mutant in which the entire coding region is deleted shows a considerable decrease in alpha-factor production and a 75% decrease in mating. Mutations in mf alpha 2 have little or no effect on alpha-factor production or mating. The mf alpha 1 mf alpha 2 double mutants are completely defective in mating and alpha-factor production. These results indicate that at least one alpha-factor structural gene product is required for mating in MAT alpha cells, that MF alpha 1 is responsible for the majority of alpha-factor production, and that MF alpha 1 and MF alpha 2 are the only active alpha-factor genes.
Base Sequence↗
Metabolism of alpha-factor by a mating type cells of Saccharomyces cerevisiae.
When a mating type cells of Saccharomyces cerevisiae are exposed to the mating pheromone alpha-factor in liquid cultures, there is a time-dependent loss of alpha-factor activity from the culture fluid. This loss of biological activity can be directly correlated with the proteolysis of the pheromone by a mating type cells. The metabolism of alpha-factor by a mating type cells may be measured by using either in vitro 125I-labeled or in vivo 35S-labeled pheromone. Addition of chloroquine to growing cultures of a mating type cells at concentrations which cause no detectable alterations in cell growth produces a potentiation of alpha-factor mediated cell cycle arrest. This potentiation of alpha-factor activity is directly correlated with the inhibition of alpha-factor proteolysis. Thus, while proteolytic digestion of alpha-factor appears to be related to the mechanism whereby a mating type cells "detoxify" alpha-factor and recover from cell cycle arrest, proteolysis of the mating factor is not necessary for alpha-factor mediated cell cycle arrest.
Chloroquine↗
Functional complementation of yeast ste6 by a mammalian multidrug resistance mdr gene.
Multidrug resistance in mammalian tumor cells is associated with the overexpression of mdr genes encoding P-glycoproteins, which function as drug efflux pumps. A yeast homolog of mdr, STE6, mediates export of a-factor mating peptide. Yeast MATa cells carrying a ste6 deletion produce no extracellular a-factor and therefore are defective in mating. Expression of a complementary DNA for the mouse mdr3 gene in a yeast ste6 deletion strain restored ability to export a-factor and to mate. A mutation (a serine to phenylalanine substitution at amino acid 939) known to affect the activity of the mdr3 gene product abolished its ability to complement the yeast ste6 deletion. Thus, functions of P-glycoproteins in normal mammalian cells may include the transmembrane export of endogenous peptides.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Roles of Wee1 and Nim1 protein kinases in regulating the switch from mitotic division to sexual development in Schizosaccharomyces pombe.
In self-fertile strains of the fission yeast Schizosaccharomyces pombe, nitrogen starvation initiates a program of sexual development in which cells express mating pheromones and receptors, arrest cell cycle progression in G1, and conjugate. This process is dependent on Rum1, an inhibitor of the Cdc2-Cdc13 and Cdc2-Cig2 cyclin B kinases. The M-phase induction activity of Cdc2-Cdc13 is inhibited by Wee1 tyrosine kinase, which phosphorylates Cdc2 on tyrosine-15. We report here that Wee1 activity is also important for mating. This discovery arose from studies of Nim1, a kinase which promotes mitosis by inhibiting Wee1. Nim1 was previously thought to have an important role in promoting mitosis during nitrogen starvation, but our studies revealed that Nim1 protein drops to an undetectable level within 15 min of nitrogen depletion. In contrast, Wee1 remains abundant, and tyrosine-phosphorylated Cdc2 is detected for at least 4 h after resuspension of cells in nitrogen-free medium. This suggested that maintenance of Wee1 activity may be important during the early stages of nitrogen starvation, a proposal confirmed by the observation that mating efficiency is reduced ca. fivefold in wee1- cells. Transcriptional induction of genes encoding mating factors and receptors is also delayed in wee1- cells. The wee1- mating defect is suppressed by deletion of cig2+, which encodes a B-type cyclin that promotes the onset of S and inhibits conjugation. These findings indicate that Wee1 and Rum1 act jointly to inhibit Cdc2 and promote sexual development in nitrogen-starved cells.
Ammonium Chloride↗
Probing the functional conformation of the tridecapeptide mating pheromone of Saccharomyces cerevisiae through study of disulfide-constrained analogs.
Analogs of the Saccharomyces cerevisiae alpha-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys7 and Gln10 were replaced with Cys, Cys(CH3), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. Cyclo7,10[Cys7,X9,Cys10,Nle12]alpha-factor , where X=D-Val, D-Ala, L-Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20-30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys7 and Gln10 with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5-10-fold more active than their sulfhydryl-containing homologs, and cyclo7,10[Cys7,L-Ala9,Cys10,Nle12] alpha-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH3) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the alpha-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form beta-turns spanning residues 7-10 than their less active linear counterparts. The results provide strong evidence that this beta-turn is important for optimal signal transduction by alpha-factor.
Amino Acid Sequence↗
Synthesis of S-alkyl and C-terminal analogs of the Saccharomyces cerevisiae a-factor. Influence of temperature on the stability of Fmoc and OFm groups toward HF.
The a-mating factor of Saccharomyces cerevisiae Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(farnesyl)OCH3, and 10 analogs modified at the cysteine side chain and/or the terminal carboxyl were synthesized using a combination of solid phase and solution phase methodologies. The strategy of synthesis involved the condensation of an amine terminal protected decapeptide with a carboxyl terminal S-alkylated dipeptide ester or amide using benzotriazol-l-yloxy-tris(methylamino)-phosphonium hexafluorophosphate as the coupling agent. The protected decapeptide was assembled on a PAM-resin using 9-fluorenylmethoxycarbonyl (Fmoc) for the protection of the Tyr alpha-amine and Lys epsilon-amine and 9-fluorenylmethyl ester (OFm) for the protection of the Asp beta-carboxyl. Premature loss of the OFm group from the HF cleavage was observed at 0-2 degrees, whereas no loss occurred when the cleavage reaction was conducted at -5 degrees. In contrast to these results, the OFm group in Asp(OFm) was partially removed by HF at -5 degrees and was completely stable to HF only at -20 degrees. The S-alkylated dipeptide esters were prepared, in yields from 64% to 88%, via thioalkylation of amine protected or unprotected dipeptide esters using potassium fluoride dihydrate as the base. The use of a tertiary amine as the base of thiohexadecanylation resulted in low reactivity.
Alkylation↗
Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells.
The production of milligram quantities of purified, active, folded membrane protein from heterologous expression systems remains a general challenge due to intrinsically low expression levels, misfolding, and instability. Here we report the overexpression and purification of milligram quantities of functional Saccharomyces cerevisiae G-protein-coupled receptor, Ste2p, from transiently transfected human embryonic kidney 293 EBNA1 cells. Fluorescent microscopy indicates localization of Ste2p-GFP and Fc-Ste2p-GFP fusion receptors to the cell membrane. Up to 2 mg (approximately 10 pmol/million cells) of the Fc-Ste2p-GFP fusion and 1 mg of a Ste2p-Strep-TagII/(His)8-tagged version were purified per liter of culture following protein A-Sepharose and Talon metal affinity chromatography, respectively. Two distinct fluorescent labels, the hydrophobic 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) and the more hydrophilic fluorescein-5-maleimide (FM), were individually attached to the C-terminus of the alpha-mating factor ligand by addition of a reactive cysteine residue to produce active fluorescent pheromones. In vitro fluorescent ligand binding assays demonstrated that a high percentage of the recombinant purified receptor is correctly folded and able to bind ligand. KD values of 34 +/- 3 and 300 +/- 20 nM were observed respectively for the CPM- and FM-labeled ligands. These results combined with blue-shifted emission peaks and loss of fluorescent quenching observed for both fluorescent-labeled Cys alpha-factors when bound to receptor support a model in which the C-terminus of the ligand is packed in a hydrophobic pocket at the interface between the transmembrane and extracellular loop domains. Overall, we present an efficient system for recombinant production of milligram quantities of purified Ste2p in a biologically active form with applications to future structure and functional studies.
Cells, Cultured↗
Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae.
Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa. Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction. Production levels are relatively low (ca. 1 mg/liter). Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting. The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS. From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca. 1% of the purified r-ATS), were characterized. Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form. RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing. Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system. Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay. Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide. The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.
Amino Acid Sequence↗
Engineering of Pichia pastoris for improved production of antibody fragments.
The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins, including antibody fragments. However, limitations became obvious especially when secreting heterodimeric Fab fragments. Up-to-date, antibody fragments have only been expressed under control of the strong inducible alcohol oxidase 1 (AOX1) promoter, which may stress the cells by excessive transcription. Here, we examined the secretion characteristics of single chain and Fab fragments of two different monoclonal anti-HIV1 antibodies (2F5 and 2G12) with both the AOX1 and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Also, the influences of different secretion leaders and strains were evaluated. Interestingly, secretion was only achieved when using the GAP promoter and the Saccharomyces cerevisiae mating factor alpha (MFalpha leader), whereas there was no difference between the two P. pastoris strains. During fed batch fermentation of a 2F5 Fab expressing strain, intracellular retention of Fab heavy chains was observed, while both intact Fab and single light chain molecules were only detected in the supernatants. This led to the conclusion that protein folding and heterodimer assembly in the ER are rate limiting steps in Fab secretion. To alleviate this limitation, S. cerevisiae protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription factor HAC1 were constitutively overexpressed in P. pastoris. While the overexpression of HAC1 led to a moderate increase of Fab secretion of 1.3-fold, PDI enabled an increase of the Fab level by 1.9-fold. Hence, the formation of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly and subsequent secretion.
Alcohol Oxidoreductases↗
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.
Alleles↗
Arabidopsis thaliana cDNA isolated by functional complementation shows homology to serine/threonine protein kinases.
The yeast Saccharomyces cerevisiae ste6 mutant is defective in transport of a-mating factor, resulting in an inability of ste6 a cells to mate with alpha cells. The gene encodes an ATP-binding cassette, ABC transporter. We used functional complementation of a yeast ste6 mutant with an Arabidopsis thaliana expression library in an attempt to clone an Arabidopsis homolog. Sequence analysis of the isolated Arabidopsis complementing cDNA however showed no homology to the STE6 gene. High sequence similarity was detected to members of the mitogen-activated serine/threonine protein (MAP) kinase family involved in signal transduction: STE20, STE11, BCK1, Byr2 and p65PAK. The Arabidopsis clone failed to complement a fus3/kss1 mutant of S. cerevisiae, but did complement a defect in ste11, ste20, as well as ste6. The isolated clone encodes a protein that is truncated at its amino-terminal, and might function in a similar way as a dominant STE11 truncation allele. These results suggest that the Arabidopsis cDNA encodes a putative serine/threonine kinase that can function in the mating response pathway upstream of FUS3/KSS1 in S. cerevisiae, at the level of STE11 gene. Interestingly, this clone is able to restore the ability of the ste6 yeast mutant to export a-factor.
ATP-Binding Cassette Transporters↗
Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe.
Loss of function of the Schizosaccharomyces pombe gap1 gene results in the same phenotypes as those caused by an activated ras1 mutation, i.e., hypersensitivity to the mating factor and inability to perform efficient mating. Sequence analysis of gap1 indicates that it encodes a homolog of the mammalian Ras GTPase-activating protein (GAP). The predicted gap1 gene product has 766 amino acids with relatively short N- and C-terminal regions flanking the conserved core sequence of GAP. Genetic analysis suggests that S. pombe Gap1 functions primarily as a negative regulator of Ras1, like S. cerevisiae GAP homologs encoded by IRA1 and IRA2, but is unlikely to be a downstream effector of the Ras protein, a role proposed for mammalian GAP. Thus, Gap1 and Ste6, a putative GDP-GTP-exchanging protein for Ras1 previously identified, appear to play antagonistic roles in the Ras-GTPase cycle in S. pombe. Furthermore, we suggest that this Ras-GTPase cycle involves the ra12 gene product, another positive regulator of Ras1 whose homologs have not been identified in other organisms, which could function either as a second GDP-GTP-exchanging protein or as a factor that negatively regulates Gap1 activity.
Amino Acid Sequence↗
Mutational analysis of the role of N-glycosylation in alpha-factor receptor function.
The alpha-factor mating pheromone receptor (encoded by STE2) activates a G protein signaling pathway that stimulates the conjugation of Saccharomyces cerevisiae yeast cells. The alpha-factor receptor is known to undergo several forms of post-translational modification, including phosphorylation, mono-ubiquitination, and N-linked glycosylation. Since phosphorylation and mono-ubiquitination have been shown previously to play key roles in regulating the signaling activity and membrane trafficking of the alpha-factor receptors, the role of N-linked glycosylation was investigated in this study. The Asn residues in the five consensus sites for N-linked glycosylation present in the extracellular regions of the receptor protein were mutated to prevent carbohydrate attachment at these sites. Mutation of two sites near the receptor N-terminus (N25Q and N32Q) diminished the degree of receptor glycosylation, and the corresponding double mutant was not detectably N-glycosylated. The nonglycosylated receptors displayed normal function and subcellular localization, indicating that glycosylation is not important for wild-type receptor activity. However, mutation of the glycosylation sites resulted in improved plasma membrane localization for the Ste2-3 mutant receptors that are normally retained intracellularly at elevated temperatures. These results suggest that N-glycosylation may be involved in the sorting process for misfolded Ste2 proteins, and may similarly affect certain mutant receptors whose altered trafficking is implicated in human diseases.
Amino Acid Sequence↗
Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system.
A homologous cell-free system has been derived from the yeast Saccharomyces cerevisiae that allows the translation, translocation, and glycosylation of the precursors of yeast mating factor alpha and invertase. The precursors were translated in a yeast lysate from mRNA obtained by in vitro transcription of the MF alpha 1 and SUC2 genes. Inclusion of yeast microsomes resulted in the glycosylation of the alpha-factor precursor, which was demonstrated to be sequestered within the membrane vesicles. Similar results, including signal sequence cleavage, were observed for invertase. Processing of secretory proteins translated in a yeast lysate could not be achieved using microsomes derived from canine pancreas, nor were yeast microsomes active in a wheat germ translation system.
Biological Transport↗
RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins.
In the yeast Saccharomyces cerevisiae, mutations in either of two unlinked genes, RAM1 or RAM2, abolish the farnesyltransferase activity responsible for prenylation of Ras proteins and the a-factor mating pheromone. Here we report that the function of RAM1 and RAM2 genes is required for the membrane localization of Ras proteins and a-factor. The RAM2 gene was sequenced and can encode a 38-kDa protein. We examined the functional interaction of RAM2 and RAM1 by expressing the genes in Escherichia coli. Extracts derived from an E. coli strain that coexpressed RAM1 and RAM2 efficiently farnesylated a-factor peptide and Ras protein substrates. In contrast, extracts derived from E. coli strains that expressed either RAM gene alone were devoid of activity; however, when the latter extracts were mixed, protein farnesyltransferase activity was reconstituted. These results indicate that the yeast farnesyl-protein transferase is comprised of Ram1 and Ram2 polypeptides. Although Ram1 is a component of the enzyme, disruption of the RAM1 gene in yeast was not lethal, indicating that the Ram1-Ram2 farnesyltransferase is not essential for viability. In contrast, disruption of RAM2 was lethal, suggesting that Ram2 has an essential function in addition to its role with Ram1 in protein farnesylation.
Alkyl and Aryl Transferases↗
Specific induction of Ca2+ transport activity in MATa cells of Saccharomyces cerevisiae by a mating pheromone, alpha factor.
Incubation with a high concentration of a mating pheromone, alpha factor, of Saccharomyces cerevisiae induced the accumulation of Ca2+ ion in MATa cells, but not in MAT alpha or MATa/alpha cells, after a lag of 30-40 min. The alpha factor did not cause a nonspecific lesion of the membrane barrier, but induced Ca2+ transport activity specifically. This induction of Ca2+ transport activity correlated with formation of a projection on the cells, or with localized cell elongation, but not with G1 arrest or agglutinin induction. The increased Ca2+ transport activity was maintained only in the continuous presence of a high concentration of alpha factor and de novo protein synthesis. Kinetic studies of induction of Ca2+ transport by alpha factor and effects of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and Ca2+ ionophore, A23187, on mating suggested an essential role of this physiological reaction in the initiation of sexual conjugation.
Animals↗