Interactions between the ankyrin repeat-containing protein Akrlp and the pheromone response pathway in Saccharomyces cerevisiae

ABSTRACTAmong the hemiascomycetes, onlyCandida albicansmust switch from the white phenotype to the opaque phenotype to mate. In the recent evolution of this transition, mating-incompetent white cells acquired a unique response to mating pheromone, resulting in the formation of a white cell biofilm that facilitates mating. All of the upstream components of the white cell response pathway so far analyzed have been shown to be derived from the ancestral pathway involved in mating, except for the mitogen-activated protein (MAP) kinase scaffold protein, which had not been identified. Here, through binding and mutational studies, it is demonstrated that in both the opaque and the white cell pheromone responses, Cst5 is the scaffold protein, supporting the evolutionary scenario proposed. Although Cst5 plays the same role in tethering the MAP kinases as Ste5 does inSaccharomyces cerevisiae, Cst5 is approximately one-third the size and has only one rather than four phosphorylation sites involved in activation and cytoplasmic relocalization.IMPORTANCECandida albicansmust switch from white to opaque to mate. Opaque cells then release pheromone, which not only induces cells to mate but also in a unique fashion induces mating-incompetent white cells to form biofilms that facilitate opaque cell mating. All of the tested upstream components of the newly evolved white cell pheromone response pathway, from the receptor to the mitogen-activated protein (MAP) kinase cascade, are the same as those of the conserved opaque cell response pathway. One key element, however, remained unidentified, the scaffold protein for the kinase cascade. Here, we demonstrate that Cst5, a homolog of theSaccharomyces cerevisiaescaffold protein Ste5, functions as the scaffold protein in both the opaque and the white cell pheromone responses. Pheromone induces Cst5 phosphorylation, which is involved in activation and cytoplasmic localization of Cst5. However, Cst5 contains only one phosphorylation site, not four as in theS. cerevisiaeortholog Ste5. These results support the hypothesis that the entire upper portion of the newly evolved white cell pheromone response pathway is derived from the conserved pheromone response pathway in the mating process.

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The Glc7p-Interacting Protein Bud14p Attenuates Polarized Growth, Pheromone Response, and Filamentous Growth in Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.1.6.884-894.2002 ◽

2002 ◽

Vol 1 (6) ◽

pp. 884-894 ◽

Cited By ~ 19

Author(s):

Paul J. Cullen ◽

George F. Sprague

Keyword(s):

Saccharomyces Cerevisiae ◽

Fluorescent Protein ◽

Genetic Selection ◽

Positive Function ◽

Filamentous Growth ◽

Polarized Growth ◽

Protein Fusion ◽

Pheromone Response ◽

Interacting Protein ◽

Pheromone Response Pathway

ABSTRACT A genetic selection in Saccharomyces cerevisiae for mutants that stimulate the mating pathway uncovered a mutant that had a hyperactive pheromone response pathway and also had hyperpolarized growth. Cloning and segregation analysis demonstrated that BUD14 was the affected gene. Disruption of BUD14 in wild-type cells caused mild stimulation of pheromone response pathway reporters, an increase in sensitivity to mating factor, and a hyperelongated shmoo morphology. The bud14 mutant also had hyperfilamentous growth. Consistent with a role in the control of cell polarity, a Bud14p-green fluorescent protein fusion was localized to sites of polarized growth in the cell. Bud14p shared morphogenetic functions with the Ste20p and Bni1p proteins as well as with the type 1 phosphatase Glc7p. The genetic interactions between BUD14 and GLC7 suggested a role for Glc7p in filamentous growth, and Glc7p was found to have a positive function in filamentous growth in yeast.

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The DAC2/FUS3 protein kinase is not essential for transcriptional activation of the mating pheromone response pathway in Saccharomyces cerevisiae

MGG Molecular & General Genetics ◽

10.1007/bf00279392 ◽

1992 ◽

Vol 235 (2-3) ◽

pp. 450-452 ◽

Cited By ~ 9

Author(s):

Hiro-aki Fujimura

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Kinase ◽

Transcriptional Activation ◽

Pheromone Response ◽

Mating Pheromone ◽

Pheromone Response Pathway

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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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Kar4p, a karyogamy-specific component of the yeast pheromone response pathway.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.3990 ◽

1996 ◽

Vol 16 (8) ◽

pp. 3990-4002 ◽

Cited By ~ 38

Author(s):

L J Kurihara ◽

B G Stewart ◽

A E Gammie ◽

M D Rose

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Fusion ◽

Regulatory Control ◽

Diploid Nucleus ◽

Pheromone Response ◽

Specific Component ◽

Pheromone Response Pathway ◽

G2 Phase ◽

Inducible Genes

Karyogamy is the process whereby two haploid nuclei fuse to form a diploid nucleus during mating in Saccharomyces cerevisiae. Here, we describe the characterization of the KAR4 gene, previously identified in a screen for new nuclear fusion-defective mutants. During mating, kar4 mutants were defective for the microtubule-dependent movement of nuclei, a phenotype identical to that of mutations in KAR3 and CIK1. Consistent with its mutant phenotype, we found that the kar4 mutation resulted in failure to induce KAR3 and CIK1 mRNA during mating. Expression of KAR3 and CIK1 under independent regulatory control suppressed the kar4 defect, indicating that KAR4 is required primarily for the induction of KAR3 and CIK1. KAR4 was also required for meiosis, during which it may regulate KAR3; however, mitotic expression of KAR3 and CIK1 during S/G2 phase was independent of KAR4. A 30-bp region upstream of KAR3 conferred both KAR4- and STE12-dependent induction by mating pheromone. This region contained one moderate and two weak matches to the consensus pheromone response element to which the Ste12p transcriptional activator binds and five repeats of the sequence CAAA(A). Overproduction of Ste12p suppressed the kar4 defect in KAR3 induction and nuclear fusion. In contrast, Ste12p-independent expression of Kar4p did not alleviate the requirement for Ste12p during KAR3 induction. We propose that Kar4p assists Ste12p in the pheromone-dependent expression of KAR3 and CIK1. KAR4 defines a novel level of regulation for the pheromone response pathway, acting at a subset of Stel2p-inducible genes required for karyogamy.

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Membrane-displayed peptide ligand activates the pheromone response pathway in Saccharomyces cerevisiae

The Journal of Biochemistry ◽

10.1093/jb/mvs027 ◽

2012 ◽

Vol 151 (5) ◽

pp. 551-557 ◽

Cited By ~ 14

Author(s):

Keisuke Hara ◽

Takuya Ono ◽

Kouichi Kuroda ◽

Mitsuyoshi Ueda

Keyword(s):

Saccharomyces Cerevisiae ◽

Peptide Ligand ◽

Pheromone Response ◽

Pheromone Response Pathway

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Regulation of postreceptor signaling in the pheromone response pathway of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3720-3726.1989 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3720-3726

Author(s):

D Blinder ◽

D D Jenness

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Division ◽

Prolonged Exposure ◽

Pheromone Response ◽

Down Regulation ◽

Constitutive Activation ◽

Pheromone Response Pathway ◽

A Cells ◽

Alpha Factor

alpha-Factor pheromone inhibits division of yeast a cells. After prolonged exposure to alpha-factor, the cells adapt to the stimulus and resume cell division. The sst2 mutation is known to inhibit adaptation. This report examines adaptation in scg1 (also designated gpa1) and STE4Hpl (Hpl indicates haploid lethal) mutants that exhibit constitutive activation of the pheromone response pathway. Recovery of the STE4Hpl mutant was blocked by the sst2-1 mutation, whereas recovery of the scg1-7 mutant was not completely blocked by sst2-1. These results indicate that both SST2-dependent and -independent mechanisms regulate postreceptor events in the pheromone response pathway. Down regulation of receptors in response to alpha-factor was independent of the signal that was generated in the scg1 mutant.

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Pheromone response elements are necessary and sufficient for basal and pheromone-induced transcription of the FUS1 gene of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.2952-2961.1991 ◽

1991 ◽

Vol 11 (6) ◽

pp. 2952-2961 ◽

Cited By ~ 3

Author(s):

D C Hagen ◽

G McCaffrey ◽

G F Sprague

Keyword(s):

Saccharomyces Cerevisiae ◽

Single Copy ◽

Upstream Region ◽

Lacz Reporter ◽

Pheromone Response ◽

Lacz Reporter Gene ◽

Pheromone Response Pathway ◽

Yeast Genes ◽

Necessary And Sufficient ◽

Activation Sequence

The FUS1 gene of Saccharomyces cerevisiae is transcribed in a and alpha cells, not in a/alpha diploids, and its transcription increases dramatically when haploid cells are exposed to the appropriate mating pheromone. In addition, FUS1 transcription is absolutely dependent on STE4, STE5, STE7, STE11, and STE12, genes thought to encode components of the pheromone response pathway. We now have determined that the pheromone response element (PRE), which occurs in four copies within the FUS1 upstream region, functions as the FUS1 upstream activation sequence (UAS) and is responsible for all known aspects of FUS1 regulation. In particular, deletion of 55 bp that includes the PREs abolished all transcription, and a 139-bp fragment that includes the PREs conferred FUS1-like expression to a CYC1-lacZ reporter gene. Moreover, three or four copies of a synthetic PRE closely mimicked the activity conferred by the 139-bp fragment, and even a single copy of PRE conferred a trace of activity that was haploid specific and pheromone inducible. In the FUS1 promoter context, four copies of the synthetic PRE inserted at the site of the 55-bp deletion restored full FUS1 transcription. Sequences upstream and downstream from the PRE cluster were important for maximal PRE-directed expression but, by themselves, did not have UAS activity. Other yeast genes with PREs, e.g., STE2 and BAR1, are more modestly inducible and have additional UAS elements contributing to the overall activity. In the FUS1 promoter, the PREs apparently act alone to confer activity that is highly stimulated by pheromone.

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