Degradation of a-factor by a Saccharomyces cerevisiae alpha-mating-type-specific endopeptidase: evidence for a role in recovery of cells from G1 arrest.

Mating response between opposite mating types of Saccharomyces cerevisiae is dependent upon alpha factor, a tridecapeptide, and a-factor, an isoprenylated, methyl esterified dodecapeptide whose interaction with the alpha target cell has not been characterized. We report on the first biochemical and physiological evidence of an alpha-mating-type-specific a-factor-degrading activity. Radioiodinated a-factor was used to identify the a-factor-degrading activity, which is cell associated, endoproteolytic, and not required for response to pheromone. a-factor degradation was not energy dependent, nor did it require pheromone internalization or interaction with its receptor. Phenylmethylsulfonyl fluoride and tosyl-L-arginyl-methyl ester inhibited degradation of a-factor and increased the time required by alpha cells to recover from a-factor-induced growth arrest and morphological alteration, providing evidence that a-factor degradation plays a role in the recovery of alpha cells from the pheromone response.

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Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones.

Molecular and Cellular Biology ◽

10.1128/mcb.2.1.11 ◽

1982 ◽

Vol 2 (1) ◽

pp. 11-20 ◽

Cited By ~ 208

Author(s):

R K Chan ◽

C A Otte

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Solid Medium ◽

G1 Arrest ◽

Alpha Cells ◽

Opposite Mating Type ◽

Alpha Factor ◽

Complementation Groups ◽

Chromosome Iii ◽

The Right

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.

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Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones

Molecular and Cellular Biology ◽

10.1128/mcb.2.1.11-20.1982 ◽

1982 ◽

Vol 2 (1) ◽

pp. 11-20

Author(s):

R K Chan ◽

C A Otte

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Solid Medium ◽

G1 Arrest ◽

Alpha Cells ◽

Opposite Mating Type ◽

Alpha Factor ◽

Complementation Groups ◽

Chromosome Iii ◽

The Right

Eight independently isolated mutants which are supersensitive (Sst-) to the G1 arrest induced by the tridecapeptide pheromone alpha factor were identified by screening mutagenized Saccharomyces cerevisiae MATa cells on solid medium for increased growth inhibition by alpha factor. These mutants carried lesions in two complementation groups, sst1 and sst2. Mutations at the sst1 locus were mating type specific: MATa sst1 cells were supersensitive to alpha factor, but MAT alpha sst1 cells were not supersensitive to a factor. In contrast, mutations at the sst2 locus conferred supersensitivity to the pheromones of the opposite mating type on both MATa and MAT alpha cells. Even in the absence of added alpha pheromone, about 10% of the cells in exponentially growing cultures of MATa strains carrying any of three different alleles of sst2 (including the ochre mutation sst2-4) had the aberrant morphology ("shmoo" shape) that normally develops only after MATa cells are exposed to alpha factor. This "self-shmooing" phenotype was genetically linked to the sst2 mutations, although the leakiest allele isolated (sst2-3) did not display this characteristic. Normal MATa/MAT alpha diploids do not respond to pheromones; diploids homozygous for an sst2 mutation (MATa/MAT alpha sst2-1/sst2-1) were still insensitive to alpha factor. The sst1 gene was mapped to within 6.9 centimorgans of his6 on chromosome IX. The sst2 gene was unlinked to sst1, was not centromere linked, and was shown to be neither linked to nor centromere distal to MAT on the right arm of chromosome III.

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Multiple regulation of STE2, a mating-type-specific gene of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2106-2114.1986 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2106-2114

Author(s):

A Hartig ◽

J Holly ◽

G Saari ◽

V L MacKay

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Regulation ◽

Gene Product ◽

Mating Type ◽

Type A ◽

Specific Gene ◽

Alpha Cells ◽

Pheromone Response ◽

Mating Pheromone ◽

A Cells

The Saccharomyces cerevisiae STE2 gene, which is required for pheromone response and conjugation specifically in mating-type a cells, was cloned by complementation of the ste2 mutation. Transcription of STE2 is repressed by the MAT alpha 2 gene product, so that the 1.4-kilobase STE2 RNA is detected only in a or mat alpha 2 strains, not in alpha or a/alpha cells. However, STE2 RNA levels are also increased by the mating pheromone alpha-factor and decreased in strains bearing mutations in the nonspecific STE4 gene. Regulation of STE2 expression in a cells is therefore achieved by several mechanisms.

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Multiple regulation of STE2, a mating-type-specific gene of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2106 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2106-2114 ◽

Cited By ~ 38

Author(s):

A Hartig ◽

J Holly ◽

G Saari ◽

V L MacKay

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Regulation ◽

Gene Product ◽

Mating Type ◽

Type A ◽

Specific Gene ◽

Alpha Cells ◽

Pheromone Response ◽

Mating Pheromone ◽

A Cells

The Saccharomyces cerevisiae STE2 gene, which is required for pheromone response and conjugation specifically in mating-type a cells, was cloned by complementation of the ste2 mutation. Transcription of STE2 is repressed by the MAT alpha 2 gene product, so that the 1.4-kilobase STE2 RNA is detected only in a or mat alpha 2 strains, not in alpha or a/alpha cells. However, STE2 RNA levels are also increased by the mating pheromone alpha-factor and decreased in strains bearing mutations in the nonspecific STE4 gene. Regulation of STE2 expression in a cells is therefore achieved by several mechanisms.

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Temperature-Sensitive Lethal Pseudorevertants of ste Mutations in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/115.4.627 ◽

1987 ◽

Vol 115 (4) ◽

pp. 627-636

Author(s):

Margaret E Katz ◽

Jill Ferguson ◽

Steven I Reed

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Complementation Group ◽

Temperature Sensitive ◽

G1 Arrest ◽

Pheromone Response ◽

Cycle Arrest ◽

Homogeneous Cell ◽

Complementation Groups ◽

Mutant Cells

ABSTRACT A procedure was devised to isolate mutations that could restore conjugational competence to temperature sensitive ste mutants and simultaneously confer temperature-sensitive lethal growth phenotypes. Three such mutations, falling into two complementation groups, were identified on the basis of suppression of ste5 alleles. These same mutations were later shown to be capable of suppressing ste4 and ste7 alleles. Five mutations in a single complementation group were isolated as suppressors of ste2 alleles. None of the mutations described in this study conferred a homogeneous cell cycle arrest phenotype, and all were shown to define complementation groups distinct from those previously identified in studies of cell division cycle (cdc) mutations. In no instance did pseudoreversion appear to be achieved by mutational G1 arrest of ste mutant cells. Instead, it is proposed that the mutations restore conjugation by reestablishing the normal pheromone response.

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Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2966-2972.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2966-2972

Author(s):

M de Barros Lopes ◽

J Y Ho ◽

S I Reed

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

G Protein ◽

G1 Arrest ◽

Restrictive Temperature ◽

Gene Products ◽

Pheromone Response ◽

Mating Pheromone ◽

Pheromone Response Pathway ◽

Mutational Activation

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.

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Mechanism of MAT alpha donor preference during mating-type switching of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.2.657 ◽

1996 ◽

Vol 16 (2) ◽

pp. 657-668 ◽

Cited By ~ 25

Author(s):

X Wu ◽

J K Moore ◽

J E Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Strand Break ◽

Normal Donor ◽

Alpha Cells ◽

Cis Acting ◽

Mating Type Switching ◽

Chromosome Iii ◽

The Right ◽

Donor Preference

During homothallic switching of the mating-type (MAT) gene in Saccharomyces cerevisiae, a- or alpha-specific sequences are replaced by opposite mating-type sequences copied from one of two silent donor loci, HML alpha or HMRa. The two donors lie at opposite ends of chromosome III, approximately 190 and 90 kb, respectively, from MAT. MAT alpha cells preferentially recombine with HMR, while MATa cells select HML. The mechanisms of donor selection are different for the two mating types. MATa cells, deleted for the preferred HML gene, efficiently use HMR as a donor. However, in MAT alpha cells, HML is not an efficient donor when HMR is deleted; consequently, approximately one-third of HO HML alpha MAT alpha hmr delta cells die because they fail to repair the HO endonuclease-induced double-strand break at MAT. MAT alpha donor preference depends not on the sequence differences between HML and HMR or their surrounding regions but on their chromosomal locations. Cloned HMR donors placed at three other locations to the left of MAT, on either side of the centromere, all fail to act as efficient donors. When the donor is placed 37 kb to the left of MAT, its proximity overcomes normal donor preference, but this position is again inefficiently used when additional DNA is inserted in between the donor and MAT to increase the distance to 62 kb. Donors placed to the right of MAT are efficiently recruited, and in fact a donor situated 16 kb proximal to HMR is used in preference to HMR. The cis-acting chromosomal determinants of MAT alpha preference are not influenced by the chromosomal orientation of MAT or by sequences as far as 6 kb from HMR. These data argue that there is an alpha-specific mechanism to inhibit the use of donors to the left of MAT alpha, causing the cell to recombine most often with donors to the right of MAT alpha.

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Mutations affecting donor preference during mating type interconversion in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/139.4.1495 ◽

1995 ◽

Vol 139 (4) ◽

pp. 1495-1510 ◽

Cited By ~ 2

Author(s):

K S Weiler ◽

L Szeto ◽

J R Broach

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosome Segregation ◽

Mating Type ◽

Null Allele ◽

Alpha Cells ◽

Opposite Mating Type ◽

Donor Preference ◽

Donor Locus ◽

Type Information ◽

Selection Of

Abstract Homothallic strains of Saccharomyces cerevisiae can convert mating type from a to alpha or alpha to a as often as every generation, by replacing genetic information specifying one mating type at the expressor locus, MAT, with information specifying the opposite mating type. The cryptic mating type information that is copied and inserted at MAT is contained in either of two loci, HML or HMR. The particular locus selected as donor during mating type interconversion is regulated by the allele expressed at MAT. MATa cells usually select HML, and MAT alpha cells usually select HMR, a process referred to as donor preference. To identify factors required for donor preference, we isolated and characterized a number of mutants that frequently selected the nonpreferred donor locus during mating type interconversion. Many of these mutants were found to harbor chromosome rearrangements or mutations at MAT or HML that interfered with the switching process. However, one mutant carried a recessive allele of CHL1, a gene previously shown to be required for efficient chromosome segregation during mitosis. Homothallic strains of yeast containing a null allele of CHL1 exhibited almost random selection of the donor locus in a MATa background but were normal in their ability to select HMR in a MAT alpha background. Our results indicate that Chl1p participates in the process of donor selection and are consistent with a model in which Chl1p helps establish an intrinsic bias in donor preference.

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A role for CDC7 in repression of transcription at the silent mating-type locus HMR in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1080 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1080-1091 ◽

Cited By ~ 36

Author(s):

A Axelrod ◽

J Rine

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Site ◽

Mating Type ◽

Double Mutant ◽

Temperature Sensitive ◽

Alpha Cells ◽

Nucleotide Substitutions ◽

Type Locus

The mating-type genes at MAT in Saccharomyces cerevisiae are expressed, whereas the same genes located at HML and HMR are transcriptionally repressed. The DNA element responsible for repression at HMR has been termed a silencer and contains an autonomous replication sequence, a binding site for GRFI/RAPI, and a binding site for ABFI. A double-mutant HMR-E silencer that contains single nucleotide substitutions in both the GRFI/RAPI- and ABFI-binding sites no longer binds either factor in vitro, nor represses transcription at HMR in vivo. In MAT alpha cells, this derepression of a information results in a nonmating phenotype. Second-site suppressor mutations were isolated that restored the alpha mating phenotype to MAT alpha cells containing the double-mutant silencer. One of these suppressors, designated sas1-1, conferred a temperature-sensitive lethal phenotype to the cell. SAS1 was found to be identical to CDC7, a gene which encodes a protein kinase required for the initiation of DNA replication. This new allele of CDC7 was designated cdc7-90. cdc7-90 restored the alpha mating phenotype by restoring silencing. The original allele of CDC7, isolated on the basis of the cell cycle phenotype it confers, also restored silencing, and overexpression of CDC7 interfered with silencing. cdc7-90 did not restore detectable binding of GRFI/RAPI or ABFI to the double-mutant silencer in vitro. These results indicate that a reduced level of CDC7 function restores silencing to a locus defective in binding two factors normally required for silencing.

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