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Results for “Receptors, Mating Factor”

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PubMed2000

The Leu-132 of the Ste4(Gbeta) subunit is essential for proper coupling of the G protein with the Ste2 alpha factor receptor during the mating pheromone response in yeast.

In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered alpha pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the beta propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and alpha cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromone-stimulated receptor and that this domain could be an important point for physical interaction between the Gbeta and the Galpha subunits.

Amino Acid Substitution↗

PubMed1991

Isolation and characterization of a gene encoding a G-protein alpha subunit from Schizosaccharomyces pombe: involvement in mating and sporulation pathways.

The gpal gene of Schizosaccharomyces pombe, which encodes a protein homologous with the alpha subunits of mammalian guanine nucleotide-binding proteins (G proteins), was isolated by cross-hybridization using rat Gi1 alpha and Gx alpha cDNA. The deduced amino acid sequence was about 37% identical with rat Gi1 alpha and Gx alpha proteins and contained three conserved motifs commonly found in all GTP-binding proteins. Disruption of gpa1 was not lethal but conferred sterility and sporulation deficiency on Sch. pombe cells. Thus, the gene is essential for the sexual development and is probably coupled to mating-factor receptors. In contrast to Saccharomyces cerevisiae GPA1, which plays a negative role in mating-factor signal transduction, Sch. pombe gpa1+ apparently has a positive function. A gpa1 transcript of 2.2 kilobases was detected in vegetatively growing cells. A 1.6-kilobase gpa1 transcript appeared in addition to the 2.2-kilobase transcript when cells were derepressed for mating or meiosis.

Amino Acid Sequence↗

PubMed2003

Aromatic residues at the extracellular ends of transmembrane domains 5 and 6 promote ligand activation of the G protein-coupled alpha-factor receptor.

The alpha-factor receptor (STE2) stimulates a G protein signaling pathway that promotes mating of the yeast Saccharomyces cerevisiae. Previous random mutagenesis studies implicated residues in the regions near the extracellular ends of the transmembrane domains in ligand activation. In this study, systematic Cys scanning mutagenesis across the ends of transmembrane domains 5 and 6 identified two residues, Phe(204) and Tyr(266), that were important for receptor signaling. These residues play a specific role in responding to alpha-factor since the F204C and Y266C substituted receptors responded to an alternative agonist (novobiocin). To better define the structure of this region, the Cys-substituted mutant receptors were assayed for reactivity with a thiol-specific probe that does not react with membrane-imbedded residues. A drop in reactivity coincided with residues likely to be buried in the membrane. Interestingly, both Phe(204) and Tyr(266) are located very near the interface region. However, these assays predict that Phe(204) is accessible at the surface of the receptor, consistent with the strong defect in binding alpha-factor caused by mutating this residue. In contrast, Tyr(266) was not accessible. This correlates with the ability of Y266C mutant receptors to bind alpha-factor and suggests that this residue is involved in the subsequent triggering of receptor activation. These results highlight the role of aromatic residues near the ends of the transmembrane segments in the alpha-factor receptor, and suggest that similar aromatic residues may play an important role in other G protein-coupled receptors.

Amino Acid Sequence↗

PubMed1999

Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae.

The alpha-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the alpha-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein alpha subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product.

Amino Acid Sequence↗

PubMed2000

The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes to the formation of preactivation complexes with its cognate G protein.

Binding of the alpha-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Galpha subunits in an alpha-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Galpha subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the alpha-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for alpha-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the alpha-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor-G-protein preactivation complexes.

Alleles↗

PubMed1996

Ubiquitination of the yeast a-factor receptor.

The a-factor receptor (Ste3p) is one of two pheromone receptors in the yeast Saccharomyces cerevisiae that enable the cell-cell communication of mating. In this report, we show that this receptor is subject to two distinct covalent modifications-phosphorylation and ubiquitination. Phosphorylation, evident on the unstimulated receptor, increases upon challenge by the receptor's ligand, a-factor. We suggest that this phosphorylation likely functions in the adaptive, negative regulation of receptor activity. Removal of phosphorylation by phosphatase treatment uncovered two phosphatase-resistant modifications identified as ubiquitination using a myc-epitope-tagged ubiquitin construct. Ste3p undergoes rapid, ligand-independent turnover that depends on vacuolar proteases and also on transport of the receptor from surface to vacuole (i.e., endocytosis) (Davis, N.G., J.L.Horecka, and G.F. Sprague, Jr., 1993 J. Cell Biol. 122:53-65). An end4 mutation, isolated for its defect in the endocytic uptake of alpha-factor pheromone (Raths, S., J. Rohrer, F. Crausaz, and H. Riezman. 1993. J. Cell Biol. 120:55-65), blocks constitutive endocytosis of the a-factor receptor, yet fails to block ubiquitination of the receptor. In fact, both phosphorylation and ubiquitination of the surfacebound receptor were found to increase, suggesting that these modifications may occur normally while the receptor is at the cell surface. In a mutant strain constructed to allow for depletion of ubiquitin, the level of receptor ubiquitination was found to be substantially decreased. Correlated with this was an impairment of receptor degradative turnover-receptor half-life that is normally approximately 20 min at 30 degrees C was increased to approximately 2 h under these ubiquitin-depletion conditions. Furthermore, surface residency, normally of short duration in wild-type cells (terminated by endocytosis to the vacuole), was found to be prolonged; the majority of the receptor protein remained surface localized fully 2 h after biosynthesis. Thus, the rates of a-factor receptor endocytosis and consequent vacuolar turnover depend on the available level of ubiquitin in the cell. In cells mutant for two E2 activities, i.e., ubc4 delta ubc5 delta cells, the receptor was found to be substantially less ubiquitinated, and in addition, receptor turnover was slowed, suggesting that Ubc4p and Ubc5p may play a role in the recognition of the receptor protein as substrate for the ubiquitin system. In addition to ligand-independent uptake, the a-factor receptor also undergoes a ligand-dependent form of endocytosis (Davis, N.G., J.L. Horecka, and G.F. Sprague, Jr. 1993. J. Cell. Biol. 122:53-65). Concurrent with ligand-dependent uptake, we now show that the receptor undergoes ligand-induced ubiquitination, suggesting that receptor ubiquitination may function in the ligand-dependent endocytosis of the a-factor receptor as well as in its constitutive endocytosis. To account for these findings, we propose a model wherein the covalent attachment of ubiquitin to surface receptor triggers endocytic uptake.

Acid Phosphatase↗

PubMed2003

Synthesis and biophysical characterization of a multidomain peptide from a Saccharomyces cerevisiae G protein-coupled receptor.

We attached peptides corresponding to the seventh transmembrane domain (TMD7) of the alpha-mating factor receptor (Ste2p) of Saccharomyces cerevisiae to a hydrophilic, 40-residue fragment of the carboxyl terminus of this G protein-coupled receptor. Peptides corresponding to (a) the 40-residue portion of the carboxyl tail (T-40), (b) the tail plus a part of TMD7 (M7-12-T40), and (c) to the tail plus the full TMD7 (M7-24-T40) were chemically synthesized and purified. The molecular mass and primary sequence of these peptides were confirmed by mass spectrometry and tandem mass spectrometry procedures. Circular dichroism (CD) revealed that T-40 was disordered in phosphate buffer and in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-racemic-(1-glycerol)] bilayers. In contrast, M7-12-T40 and M7-24-T40 peptides were partially helical in the presence of vesicles, and difference CD spectroscopy showed that the transmembrane regions of these peptides were 42 and 94% helical, respectively. CD analysis also demonstrated that M7-24-T40 retained its secondary structure in the presence of 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-racemic-(1-glycerol)] micelles at 0.5 mm concentration. Thus, the tail and the transmembrane domain of the multidomain 64-amino acid residue peptide manifest individual conformational preferences. Measurement of tryptophan fluorescence indicated that the transmembrane domain integrated into bilayers in a manner similar to that expected for this region in the native state of the receptor. This study demonstrated that the tail of Ste2p can be used as a hydrophilic template to study transmembrane domain structure using techniques such as CD and NMR spectroscopy.

Amino Acid Sequence↗

PubMed1986

Yeast peptide pheromones, a-factor and alpha-factor, activate a common response mechanism in their target cells.

We show that in yeast the cell type specificity of pheromone response is determined solely by the species of receptor that a cell synthesizes. The two receptor-pheromone interactions are functionally interchangeable and involve the creation of a common intracellular signal. In particular, we find that provision of a-factor receptor or alpha-factor receptor in mat alpha 1 mutants, which normally do not express either receptor or any other a- or alpha-specific products, allows response to the appropriate pheromone. Moreover, provision of a-factor receptor in a cells lacking alpha-factor receptor restores mating competence to those cells. Finally, an aspect of pheromone response that is normally unique to a-factor action on alpha cells--increased transcription from the alpha-specific STE3 gene--can also be observed following alpha-factor treatment of pseudo-a cells (mat alpha 2 ste3 ste13), special mutants that respond to alpha-factor and also have an active STE3 promoter.

Genes, Fungal↗

PubMed1997

The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).

Acyltransferases↗

PubMed1994

Noncontiguous domains of the alpha-factor receptor of yeasts confer ligand specificity.

The Saccharomyces cerevisiae alpha-factor receptor has a 3400-fold higher affinity for the S. cerevisiae alpha-factor peptide (c-alpha-f) than for the Saccharomyces kluyveri alpha-factor peptide (k-alpha-f) as determined by competition for [3H] c-alpha-f binding. The S. kluyveri alpha-factor receptor has an approximately 2-fold higher affinity for k-alpha-f than for c-alpha-f. The S. kluyveri receptor gene (k-STE2) is incompletely regulated by S. cerevisiae mating type and poorly expressed on the surface of an S. cerevisiae mating type a strain. A chimeric receptor (c/k1) with amino acid residues 1-45 derived from S. cerevisiae and amino acid residues 46-427 from S. kluyveri exhibits the binding specificity of the S. kluyveri receptor. However, chimeric receptors containing residues 1-168 (c/k2) or 1-250 (c/k3) from S. cerevisiae and the remainder from the S. kluyveri receptor exhibit specificities similar to one another, but intermediate between the parent S. cerevisiae and S. kluyveri receptors. The relative ability of c-alpha-f and k-alpha-f to induce growth arrest in strains expressing chimeric receptors parallels relative affinity. Thus, two noncontiguous domains that include putative extracellular loops 1 and 3 and associated transmembrane segments, but exclude the extracellular NH2 terminus and loop 2, appear to contribute to alpha-factor receptor ligand specificity. COOH-terminal regions of the S. kluyveri receptor appear to confer a desensitization defect when expressed in S. cerevisiae. The S. cerevisiae receptor truncated at residue 296 retains ligand specificity for growth arrest.

Amino Acid 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↗

PubMed1992

Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells.

Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.

Animals↗

PubMed1998

A function for monoubiquitination in the internalization of a G protein-coupled receptor.

Modification of an S. cerevisiae G protein-coupled receptor with ubiquitin is required for its ligand-stimulated internalization. We now demonstrate that monoubiquitination on a single lysine residue is sufficient to signal receptor internalization, a modification distinct from that required for proteasome recognition. Formation of a polyubiquitin chain is not necessary, as demonstrated by the ability of mutant ubiquitins that lack lysine residues to serve as efficient internalization signals. Fusion of ubiquitin in-frame to a receptor that lacks cytoplasmic tail lysines also promotes rapid receptor internalization, indicating that ubiquitin itself and not a specific type of linkage to the receptor acts as an internalization signal. Thus, we have defined a cellular function for monoubiquitination in alpha-factor receptor endocytosis.

Animals↗

PubMed2000

Recycling of the yeast a-factor receptor.

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway and a ligand-dependent uptake pathway. Whereas the constitutive pathway leads to degradation of the receptor in the vacuole, the present work finds that receptor internalized via the ligand-dependent pathway recycles. With the a-factor ligand continuously present in the culture medium, trafficking of the receptor achieves an equilibrium in which continuing uptake to endosomal compartments is balanced by its recycling return to the plasma membrane. Withdrawal of ligand from the medium leads to a net return of the internalized receptor back to the plasma membrane. Although recycling is demonstrated for receptors that lack the signal for constitutive endocytosis, evidence is provided indicating a participation of recycling in wild-type Ste3p trafficking as well: a-factor treatment both slows wild-type receptor turnover and results in receptor redistribution to intracellular endosomal compartments. Apparently, a-factor acts as a switch, diverting receptor from vacuole-directed endocytosis and degradation, to recycling. A model is presented for how the two Ste3p endocytic modes may collaborate to generate the polarized receptor distribution characteristic of mating cells.

Cell Fusion↗

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↗

PubMed1990

Identification, purification, and cloning of a polypeptide (PRTF/GRM) that binds to mating-specific promoter elements in yeast.

In yeast the alpha-specific regulators, alpha 1 and alpha 2 have been proposed to be DNA-binding proteins, both of which have to interact with an additional factor called PRTF or GRM, respectively, to exert their biological functions. Although the cis-acting sequence requirements for alpha 1 and alpha 2 are different, their target sequences share a common motif. PRTF or GRM is thought to act via this common DNA sequence; therefore, it has been suggested that they represent the same factor. I purified a protein that binds to this common promoter element by DNA affinity chromatography. The purified protein is able to recruit the alpha-specific activator alpha 1 to its binding sites, suggesting that it is indeed PRTF. Further evidence is presented to show that PRTF and GRM are the same protein and that PRTF plays a role in the activation of a-specific genes. Specific antibodies to the purified protein were obtained. They identify the protein as a component of DNA-protein complexes that formed with cell-type-specific promoter sequences. Using these antibodies, the gene encoding the protein was cloned from a yeast lambda gt11 expression library. The DNA sequence established that the gene encoding PRTF/GRM is identical with a previously described gene, FUN80 (essential factor of unknown function) or MCM1 (minichromosome maintenance). Sequence comparison showed further that PRTF/GRM shares similarities with a repressor from yeast, ARGRI, and the mammalian transcription factor SRF.

Amino Acid Sequence↗

PubMed1987

Common signal transduction system shared by STE2 and STE3 in haploid cells of Saccharomyces cerevisiae: autocrine cell-cycle arrest results from forced expression of STE2.

Induction of STE2 expression using the GAL1 promoter both in a wild-type MATalpha strain and in a MATalpha ste3 strain caused transient cell-cycle arrest and changes in morphology ('shmoo'-like phenotype) in a manner similar to alpha cells responding to alpha-factor. In addition, STE2 expressed in a MATalp[ha ste3 mutant allowed the cell to conjugate with alpha cells but at an efficiency lower than that of wil-type alpha cells. This result indicates that signal(s) generated by alpha-factor in alpha cells can be substituted by signal(s) generated by the interaction of alpha-factor with the expressed STE2 product. When STE2 or STE3 was expressed in a matalpha1 strain (insensitive to both alpha- and a-factors), the cell became sensitive to alpha- or a-factor, respectively, and resulted in morphological changes. These results suggest that STE2 and STE3 are the sole determinants for alpha-factor and a-factor sensitivity, respectively, in this strain. On the other hand, expression of STE2 in an a/alpha diploid cell did not affect the alpha-factor insensitive phenotype. Haploid-specific components may be necessary to transduce the alpha-factor signal. These results are consistent with the idea that STE2 encodes an alpha-factor receptor and STE3 encodes an a-factor receptor, and suggest that both alpha- and a-factors may generate an exchangeable signal(s) within haploid cells.

Cell Cycle↗

PubMed1986

Down regulation of the alpha-factor pheromone receptor in S. cerevisiae.

The peptide pheromone, alpha-factor, was found to elicit down regulation of receptor sites on yeast a cell targets. Cellular uptake of alpha-factor accompanied the loss of receptor sites. Receptor-deficient a cells bearing a deletion of the STE2 gene were unable to internalize alpha-factor. Cultures were found to reaccumulate receptor sites following the initial period of down regulation; reaccumulation was dependent upon protein synthesis. Pheromone-resistant mutants, ste4-3 and ste5-3, retained the ability to down regulate receptors but failed to show reaccumulation. Our results suggest that alpha-factor-receptor complexes enter the cell by receptor-mediated endocytosis and that receptors are continuously lost and resynthesized in the presence of alpha-factor. We found no reduction of alpha-factor binding capacity in a cell cultures that had adapted to alpha-factor.

Adaptation, Physiological↗